The role of the gut as a primary lymphoid organ: CD8αα intraepithelial T lymphocytes in euthymic mice derive from very immature CD44+ thymocyte precursors.

Intestinal CD8αα intraepithelial T lymphocytes (T-IELs) have a key role in mucosal immunity and, unlike other T cells, were proposed to differentiate locally. In apparent contradiction, these cells were also shown to originate from a wave of thymus migrants colonizing the gut in the first 3 weeks after birth. We here identify previously uncharacterized very immature CD4(-)CD8(-)CD3(-)CD44(+)CD25(int) thymocytes, which have not yet rearranged their T-cell antigen receptor (TCR), as having the capacity to leave the thymus, migrate to the blood, colonize the gut, and reconstitute CD8αα T-IEL, and show that this cell set is fully responsible for the generation of the CD8αα T-IEL pool. Thus, although the thymus may be fundamental for efficient T-cell commitment, CD8αα T-IEL' complete TCR rearrangements and TCR-αß/γδ lineage commitment must occur in the gut. These results demonstrate a major role of the gut environment as a primary lymphoid organ.

Natural killer cell-dependent apoptosis of peripheral murine hematopoietic progenitor cells in response to Fas cross-linking: involvement of tumor necrosis factor-alpha.

Recently, a marked extramedullary myelopoiesis in Fas/CD95- or FasL/CD95L-deficient mice has been reported. In the present in vitro study, the mechanisms underlying Fas-induced apoptosis of normal peripheral colony-forming unit-C (CFU-C) progenitors in the spleen were analyzed. Surprisingly, it was found that clonogenic progenitors were protected from gammaIFN plus Fas-induced programmed cell death when Lin(+) cells were removed from cultured splenocytes. The cells that rendered CFU-C sensitive to the activation of the Fas pathway did not belong to the T or the myelocytic-monocytic lineage but comprised a non-B-cell subset expressing the activation marker B220. Among CD19(-) B220(+) splenocytes, nearly half were natural killer (NK) 1.1(+) cells whose in vivo depletion or deficiency in RAG2-gamma(c)(-/-) mice abrogated the effect of Fas cross-linking. NK cells exerted their accessory function, at least in part, through tumor necrosis factor-alpha (TNF-alpha), which they readily produced during pretreatment with the anti-Fas/CD95 monoclonal antibody and IFN-gamma and whose addition could compensate for the loss of sensitivity. In conclusion, this study provides evidence that peripheral clonogenic progenitors are not directly responsive to Fas cross-linking, even in the presence of IFN-gamma, but require NK cells as a source of TNF-alpha to make them susceptible to this death pathway.

Selective involvement of the Fas (CD95)/Fas ligand pathway in bone marrow B cell progenitors.

B lymphocyte generation in bone marrow (BM) compensates for cell loses. The Fas / Fas ligand (FasL) pathway has been implicated in apoptosis of various cell types. Abnormalities of the Fas receptor or of FasL expression are associated with excessive T cell proliferation and autoimmunity. To examine the role of the Fas / FasL system in B cell differentiation, we created double-chimeric mice by transferring both C57BL / 6 (B6)-Fas(+) and lpr-FasL(+) BM cells into RAG-2(- / -) hosts. Equal numbers of stem cells were co-injected into sublethally irradiated recipients, and their progeny were studied by using antibodies directed against the B6-Ly5. 1(+)5.2(+) and lpr-Ly5.1(-)5.2(+) populations. A longitudinal study lasting for up to 6 months revealed that cells of the lpr phenotype dominated the B6 phenotype in the BM, as a result of their active proliferation. Analysis of the B cell compartment showed more lpr than B6 cells among immature HSA(hi)B220(lo) populations. In contrast, the lpr and B6 phenotypes were equally represented among mature B cells. BM transfer to second hosts indicated that B6-derived B cell progenitors were absent from the first host. These data suggest that activation of the Fas / FasL pathway disturbs the early steps of B cell development and might therefore contribute to the onset of autoimmune disorders.

Granulocyte-colony stimulating factor treatment of lupus autoimmune disease in MRL-lpr/lpr mice.

G-CSF not only functions as an endogenous hemopoietic growth factor for neutrophils, but also displays pro-Th2 and antiinflammatory properties that could be of therapeutic benefit in autoimmune settings. We evaluated the effect of treatment with G-CSF in a murine model of spontaneous systemic lupus erythematosus, a disease in which G-CSF is already administered to patients to alleviate neutropenia, a common complication. Chronic treatment of lupus-prone MRL-lpr/lpr mice with low doses (10 microg/kg) of recombinant human G-CSF, despite the induction of a shift toward the Th2 phenotype of the autoimmune response, increased glomerular deposition of Igs and accelerated lupus disease. Conversely, high-dose (200 microg/kg) treatment with G-CSF induced substantial protection, prolonging survival by >2 mo. In the animals treated with these high doses of G-CSF, neither the Th1/Th2 profile nor the serum levels of TNF-alpha and IL-10 were modified. Despite the presence of immune complexes in their kidney glomeruli, no inflammation ensued, and serum IL-12 and soluble TNF receptors remained at pre-disease levels. This uncoupling of immune complex deposition and kidney damage resulted from a local down-modulation of FcgammaRIII (CD16) expression within the glomeruli by G-CSF. Our results demonstrate a beneficial effect of high doses of G-CSF in the prevention of lupus nephritis that may hold promise for future clinical applications, provided caution is taken in dose adjustment.

Increased fetal and extramedullary hematopoiesis in Fas-deficient C57BL/6-lpr/lpr mice.

In this study, we examined the consequences of Fas deficiency on hematopoiesis in C57BL/6-lpr/lpr mice. We found a striking extramedullary increase in hematopoietic progenitor cells, comprising erythroid and nonerythroid lineages alike. These modifications preceded the lymphadenopathy, because early progenitors (colony-forming units-spleen [CFU-S] day 8) were already augmented in day-18 fetal livers of the lpr phenotype. Three weeks after birth, CFU-S increased in peripheral blood and spleen and colony-forming cells (CFU-C) began to accumulate 1 to 3 weeks later. Extramedullary myelopoiesis augmented progressively in Fas-deficient mice, reaching a maximum within 6 months. By then, mature and immature myeloid cells had infiltrated the spleen, the liver, and the peritoneal cavity. Similar changes occurred in C57BL/6-gld/gld mice, indicating that they resulted from Fas/FasL interactions. Medullary hematopoiesis was not significantly modified in adult mice of either strain. Yet, the incidence of CFU-S decreased after Fas cross-linking on normal bone marrow cells in the presence of interferon gamma, consistent with a regulatory function of Fas/FasL interactions in early progenitor cell development. These data provide evidence that Fas deficiency can affect hematopoiesis both during adult and fetal life and that these modifications occur independently from other pathologies associated with the lpr phenotype.

Involvement of the Fas (CD95) system in peripheral cell death and lymphoid organ development.

Fas-mediated apoptosis is a form of cell death that operates through a Fas-Fas ligand (FasL) interaction. In this study we investigated the role of the Fas system during development of normal and Fas-mutated lymphocytes. Irradiated RAG2-/-recipients were reconstituted with bone marrow cells from B6 and lpr mice (Fas defective) or from B6 and gld mice (FasL defective), and analyzed for long-term development. The results showed a primary role of the Fas system in peripheral cell death and thymic colonization. In the periphery, the interaction in vivo between Fas+ and Fas-T cell populations indicated that cellular homeostasis was defective. Indeed, we observed a FasL-mediated cytotoxic effect on normal-derived T cells, explaining the dominance of lpr T cells in the mixed chimeras. The Fas mutation affected neither cell activation nor cell proliferation, as the effector (Fas-) and target (Fas+) cells behaved similarly with regard to activation marker expression and cell cycle status. However, Fas-T cells failed to seed the periphery and the thymus in the long term. We suggest that this could be due to the fact that FasL is involved in the structural organization of the lymphoid compartment.

Marked depletion at the late pro-B cell stage in the bone marrow of lpr mice correlates with the development of lymphadenopathy but not autoimmunity.

We have found that old lpr mice exhibit a loss of mature B cells in the bone marrow. The deleted population is HSAlo B220hi and is generated from the peripheral pool. Abnormalities in the microenvironment could explain the absence of mature B cells. Thus, old lpr bones were grafted under the skin of normal adult Ly5.1 hosts and examined 3 weeks later for the presence of Ly5.1+ B220hi cells. Our data show that the lpr medullary compartment was efficiently restored by host B cells. These results suggest that the bone marrow microenvironment of old lpr mice is able to sustain mature B cells. However, transfer of T cell-depleted bone marrow cells from old lpr mice to Rag-2 -/- mice leads to incomplete and inefficient repopulation of the host medullary compartment. Thus, a defect at an early stage of B cell differentiation was detected: using four-color flow cytometry, we found a profound depletion of the late pro-B B220+ CD43+ HSA+ BP-1+ cell population in aging lpr mice. This depletion was not observed in old autoimmune-prone MRL-+/+ mice which develop only autoantibodies but was present in B6-lpr mice which develop a lymphadenopathy and an indolent autoimmune syndrome. Altogether, our results demonstrate an age-linked defect in the progression of B cell differentiation in lpr mice independent of the presence of autoantibodies and targeted to the late pro-B cell subset.

In vivo CD4+ lymph node T cells from lpr mice generate CD4-CD8-B220+TCR-beta low cells.

Double-negative CD4-CD8-T cells (DNT) have been shown to be the major population of T cells responsible for the massive lymphadenopathy associated with the early onset of the lupus-like syndrome in mice bearing the lpr gene. Previously, we demonstrated that these cells do not proliferate in the peripheral lymphoid organs that they invade; furthermore, we showed that a wide range of CD4 Ag expression was observed on lymph node CD4+ T cells. In this study, we used an in vivo transfer system to analyze the progeny of CD4+ T cells from B6-lpr/lpr mice. Purified CD4+ T cells injected into B6 nude mice are able to generate DNT cells; furthermore, phenotypic and functional characterizations of the DNT cells generated in vivo show that they share the same properties as DNT cells from B6-lpr/lpr mice. We also show that, after in vitro bromodeoxyuridine incorporation, only CD4+ cells cycle. From these studies, we conclude that the lymphoproliferation occurs at the CD4+ stage and that down-regulation of this Ag probably is followed by arrest of the cell cycle.

Haemopoiesis and early T-cell differentiation.

Lineage commitment is a fundamental process that is initiated during the early stages of embryogenesis. Ultimately, this leads to the generation of the humoral and cellular arms of the immune system. In a recent workshop, researchers from the diverse fields of embryology, haematology and immunology gathered to address the topic of early T-cell differentiation.

Leukaemia inhibitory factor is necessary for maintenance of haematopoietic stem cells and thymocyte stimulation.

Leukaemia inhibitory factor (LIF) has a variety of effects on different cell types in vitro, inhibiting the differentiation of embryonic stem cells and promoting the survival and/or proliferation of primitive haematopoietic precursors and primordial germ cells. Here we show that LIF-deficient mice derived by gene targeting techniques have dramatically decreased numbers of stem cells in spleen and bone marrow. Injection of spleen and marrow cells from these mice promotes long-term survival of lethally irradiated wild-type animals, however, showing that the LIF- stem cells remain pluripotent. The numbers of committed progenitors are also reduced in the spleen but not the bone marrow, suggesting that stem cells interact differently with the splenic and medullary microenvironment. Heterozygous animals are intermediate in phenotype, implying that LIF has a dosage effect, and defects in stem cell number can be compensated by exogenous LIF. LIF thus appears to be required for the survival of the normal pool of stem cells, but not their terminal differentiation.

Role of CD4+CD45RA+ T cells in the development of autoimmune diabetes in the non-obese diabetic (NOD) mouse.

The non-obese diabetic (NOD) mouse spontaneously develops a T cell-mediated autoimmune disease, sharing many features with human insulin-dependent diabetes mellitus (IDDM), leading to insulin-secreting beta cell destruction. The role of CD4+ T cells has been evidenced at two levels. First, CD4+ T cells from diabetic animals are required to transfer diabetes to non-diabetic recipients in conjunction with CD8+ effector T cells. Second, suppressive CD4+ T cells have been characterized in non-diabetic NOD mice. T cells with different functions can thus share the CD4+ phenotype. Since CD4+ T cells can be divided into at least two subgroups on the basis of CD45 isoform expression, we evaluated the distribution of CD4+ T cells expressing the CD45RA isoform on NOD mouse thymocytes and peripheral T cells. The percentage of CD45RA+ cells was dramatically increased among the most mature CD3bright thymocytes and among CD4+ T cells in lymph nodes of the NOD mouse as compared with control strains. This increase was related to the development of insulitis. Interestingly, the CD45RA isoform was expressed on most CD4+ T cells invading the islets. In vivo treatment with an anti-CD45RA mAb prevented the development of insulitis and spontaneous diabetes in female animals but not the transfer of diabetes by T cells collected from diabetic NOD donors. These results indicate that anti-CD45RA mAb is only effective if given before the full commitment of effector T cells to the destruction of islet beta cells. Thus CD4+CD45RA+ T cells play a key role in early activation steps of anti-islet immunity.

T- and B-lymphocyte differentiation potentials of spleen colony-forming cells.

Cells that generate splenic colonies within 8 days (day-8 colony-forming units-spleen [CFU-s]) are generally thought to differentiate only into erythroid/myeloid cells. The T and B lymphocyte differentiation potentials of day-8 CFU-s were evaluated and compared with those of day-12 and 5-fluorouracil (5-FU) CFU-s. This was achieved by analyzing, after intravenous and intrathymic injection, the lymphocyte progeny of cells contained within individual splenic colonies collected at day 8 and day 12 post-bone marrow cell transfer into irradiated congenic recipients. A large majority of day-8 spleen colonies generated T cells when transferred intrathymically. After intravenous (IV) injection of day-8 colonies, donor-type thymocytes emerged in 33% of the animals reconstituted with only 1 day-8 colony, but in 83% of those inoculated with a pool of 5 colonies. All post-5-FU and 75% of day-12 colonies gave rise to thymocytes after IV injection. B cells were generated by a high proportion of day-8 colonies, and by all day-12 and post 5-FU colonies. These results demonstrate that progenitors of T and B lymphocytes are generated within spleen colonies produced by at least some day-8 CFU-s and virtually all day-12 CFU-s. Whether these progenitors are CFU-s themselves or committed precursors remains an open question.

A novel CD45RA+CD4+ transient thymic subpopulation in MRL-lpr/lpr mice: its relation to non-proliferating CD4-CD8-CD45RA+ tumor cells.

MRL-lpr/lpr mice have hypertrophied lymph nodes comprising CD4-CD8- T cells. In addition, they contain CD4+CD8- T cells co-expressing the CD45RA marker. The correlation between these two subpopulations has been difficult to assess. We analyzed the expression of CD45RA (with the RA3-2C2 antibody) in various thymic and peripheral T cell subsets, using three-color immunofluorescence. We showed that in lpr mice (i) a transient CD4+CD8- thymic subset co-expresses CD45RA during the course of the disease, and (ii) thymic as well as peripheral CD4-CD8- and CD4+CD8- T cells brightly express CD45RA; furthermore (iii) in the lymph nodes, during lymphadenopathy, CD4+CD8-CD45RA+ T cells show a broad range of the CD4 fluorescence intensity, and (iv) the increase in MHC class II expression is restricted to CD45RA-T cells of the thymus and lymph nodes of lpr mice. Taken together, these data suggest that the CD4+CD8-CD45RA+ population might generate the CD4-CD8- tumor cells. In addition, using the bromodeoxyuridine labeling technique, we demonstrate that these cells are not the result of increased proliferation.

Thy-1 modulation and cell proliferation at early steps of intrathymic bone marrow cell differentiation.

Intrathymic (IT) transfer of bone marrow (BM) precursor cells in sublethally irradiated hosts has been widely used to study T cell differentiation and maturation. In this report we have used double congenic mice Ly 5.1 Thy 1.1 (host) and Ly 5.2 Thy 1.2 (donor) and detected cycling Ly 5.2+ BM cells by in vivo bromodeoxyuridine incorporation, before induction of the Thy 1.2 antigen. Until Day 9 post-transfer, some donor type cells express a high level of Thy 1.2 together with macrophage and granulocyte markers. A few days later, a Thy 1.2low population transiently B220+ was detected. Thereafter, donor type cells expressed an intermediate Thy 1.2 brightness; this population then persisted and surpassed the other subsets. Our findings permitted to establish a relationship between cell cycle and Thy 1 fluorescence intensity according to the sequence: Thy 1low resting, Thy 1low cycling, Thy 1high cycling, Thy 1high resting. Moreover, we have shown that cells from the myeloïd and B lineages can, in vivo, transiently express the Thy 1 antigen, develop and differentiate within the thymus microenvironment.

Molecular analysis of a pro-T cell clone transformed by Abelson-murine leukemia virus, displaying progressive gamma delta T cell receptor gene rearrangement and surface expression.

We present a molecular analysis of T cell differentiation in a set of clones derived from in vitro Abelson murine leukemia virus (A-MuLV) infection of fetal liver cells. The parental clone had partial rearrangement of the beta and gamma loci and spontaneously displayed progressive rearrangement of V gamma genes during in vitro culture. Further differentiation of these clones leading to delta gene rearrangement and CD4 expression, then CD8, CD3 and T cell receptor gamma delta chain surface expression was obtained after intrathymic transfer followed by in vitro co-culture with thymic tissue. These A-MuLV clones, therefore, appear to represent a powerful model system for studying the early molecular events of T cell development at the clonal level.

CD45RA antibodies split the CD3bright T cell subset.

Thymocyte subsets have been well characterized on the basis of CD4 and CD8 antigen expression. Recently, the use of anti-CD3 antibodies has allowed more precise phenotyping of these subsets. The most immature T cell precursors are largely CD3-CD4-CD8-, while the most mature are CD3brightCD4+CD8- or CD3brightCD4-CD8+. Moreover, the expression of CD45RA on thymocytes appears to define a progenitor population and may define a continuous lineage of cells. Using a panel of CD45RA antibodies, we have further characterized the CD45RA+ thymocyte population in the murine system. The size of this subset is greatly enhanced in cortisone-treated mice and in sublethally irradiated mice. Moreover, the CD45RA+ population is present early in foetal life and is maintained thereafter. Using three-colour immunofluorescence, we show that (i) while most CD45RA+ cells are present amongst the CD4-CD8- thymocyte subset in the normal thymus, after cortisone treatment or irradiation, all four thymocyte subsets co-express significant amounts of CD45RA. This suggests that not only progenitor cells but also the mature population which can survive such manipulation are CD45RA+; and (ii) a large proportion of CD45RA+ cells are CD3bright and this subset is represented in the thymus at all stages of maturation tested. These data suggest that a proportion of TCR-gamma delta + CD3+ cells in the fetus as well as of TCR-alpha beta+ CD3+ cells in the adult co-express CD45RA.

Persistence of stem cell activity within the murine thymus after transfer of a bone marrow fraction enriched in CFU-S.

Mechanisms involved in prothymocyte migration, differentiation and self-commitment were investigated. We used a murine bone marrow fraction isolated on a discontinuous Ficoll gradient and enriched 10-20 times in CFU-S activity, and studied its fate after intrathymic transfer over a period of 200 days. In order to assess their hemopoietic activity, chimeric thymuses were intravenously transferred to secondary lethally irradiated hosts and both day 8 and day 12 spleen colonies were evaluated. The results show that transfer of a stem cell enriched fraction leads to long-term repopulation of the thymuses and that the input of progenitors is regulated by the size of the intrathymic precursor pool. Furthermore, stem cells can locate within the irradiated thymus and remain in a primitive stage for several months.

A role for the thymic epithelium in the selection of pre-T cells from murine bone marrow.

A rat thymic epithelial cell line IT45-R1 has been previously described as secreting soluble molecules that in vitro chemoattract rat hemopoietic precursor cells. The development of such an in vitro migration assay was based on the ability of cells to migrate across polycarbonate filters in Boyden chambers. In the present paper, by using the same strategy, we studied murine bone marrow cells capable of migrating in vitro toward IT45-R1 conditioned medium. The responding cells were shown to represent a minor bone marrow subpopulation characterized by a low capacity to incorporate tritiated thymidine in vitro (less than 10% of control). Moreover, this cell subset was considerably impoverished with respect to granulocyte-macrophage CFU (less than 7% of control) and pluripotent hemopoietic stem cells (less than 12% of control). Potential generation of T cells of donor-type in the lymphoid organs of irradiated recipients was measured by using C57BL/Ka Thy-1.1 and Thy-1.2 congenic mice. Thy-1.1 irradiated mice were injected intrathymically or intravenously with the selectively migrated cell subset of Thy-1.2 donor-type bone marrow cells. The use of an i.v. transfer route allowed us to show that these cells possess thymus-homing and colonization abilities. In a time-course study after intrathymic cell transfer, these migrated cells were able to generate Thy-1.2+ donor-type thymocytes represented by all cortical and medullary cell subsets in a single wave of repopulation from day 20 to day 30 after transfer, with a peak around days 23 to 25. The degree of repopulation closely resembled that seen with unfractionated bone marrow cells in terms of absolute numbers of donor cells per thymus (82% of control, 22 x 10(6) Thy-1.2+ cells) as well as in percent donor cells per thymus (105% of control). Thy-1.2+ cells were also detected in the lymph nodes and the spleens of reconstituted recipient mice. Taken together, these results support the idea that the supernatant of the established thymic epithelium IT45-R1 induces the migration of a murine bone marrow subset that contains hemopoietic stem cells already committed to the lymphoid lineage (i.e., pre-T cells).

Cell proliferation and thymocyte subset reconstitution in sublethally irradiated mice: compared kinetics of endogenous and intrathymically transferred progenitors.

After sublethal (6 Gy) whole-body irradiation, the C57BL/Ba (Thy-1.1) murine thymus regenerated in two waves, on days 3-10 and 25-32, separated by a severe relapse. The second phase of depletion-reconstitution reproduced the first one, in a less synchronous manner. The depletion affected all cell subsets, but CD4+ CD8- cells decreased later than immature cells. Cell proliferation, measured by BrdUrd incorporation, started on day 3 after irradiation and concerned CD4- CD8-, CD4- CD8+, and CD4+ CD8+ cells, sequentially. CD4+ CD8- cells never represented a significant percentage of cycling cells. When irradiation was immediately followed by an intrathymic injection of 10(5) C57BL/Ka (Thy-1.2) bone marrow cells, the relapse in thymus reconstitution was no longer observed. Detected with anti-Thy-1.2 antibodies, donor cells started cycling on day 14 and showed only one wave of proliferation. In these chimeras, recipient thymocytes behave exactly like thymocytes of solely irradiated mice. Intrathymically transferred CD4- CD8- thymocytes (10(5] showed the same proliferation kinetics as endogenous cells, with a peak in number on day 10 but completely disappeared from the thymus on days 14-21. These data reflect maturational differences between intrathymic and bone marrow precursor cells and suggest different radiosensitivities not linked to proliferative status. The resting state of the thymus immigrants was shown by the absence of Thy-1 acquisition by bone marrow cells continuously labeled for 10 days with BrdUrd in vivo before intrathymic transfer. When such labeled bone marrow cells were injected in the thymus, only the minor BrdUrd- subset gave rise to Thy-1+ cells.

Repopulation potential of thymocytes forming rosettes with phagocytic cells of the thymic reticulum.

Thymocytes binding in vitro to phagocytic cells of the thymic reticulum (P-TR), termed 'rosetting thymocytes', were injected intravenously into irradiated congenic mice and their migration patterns were compared with those that do not bind to P-TR, called 'non-rosetting thymocytes', similarly transferred. Donor cells, C57BL/Ka Thy 1.2, were distinguished from recipient cells, C57BL/Ka Thy 1.1 by a direct immunofluorescence technique using an anti-Thy 1.2 monoclonal antibody. The results demonstrate that the rosetting thymocytes have a greater capacity for homing back to the thymus and for populating the mesenteric lymph node and the spleen. Intrathymic transfer assay revealed that the donor-derived cells detected in the peripheral organs were of thymic origin.