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Extrathymic T cell precursors can be detected in many tissues and represent an immediately competent population for rapid T cell reconstitution in the event of immunodeficiencies. Blood T cell progenitors have been detected, but their source in the bone marrow (BM) remains unclear. Prospective purification of BM-resident and circulating progenitors, together with RT-PCR single-cell analysis, was used to evaluate and compare multipotent progenitors (MPPs) and common lymphoid progenitors (CLPs). Molecular analysis of circulating progenitors in comparison with BM-resident progenitors revealed that CCR9(+) progenitors are more abundant in the blood than CCR7(+) progenitors. Second, although Flt3(-) CLPs are less common in the BM, they are abundant in the blood and have reduced Cd25(+)-expressing cells and downregulated c-Kit and IL-7Rα intensities. Third, in contrast, stage 3 MPP (MPP3) cells, the unique circulating MPP subset, have upregulated Il7r, Gata3, and Notch1 in comparison with BM-resident counterparts. Evaluation of the populations' respective abilities to generate splenic T cell precursors (Lin(-)Thy1.2(+)CD25(+)IL7Rα(+)) after grafting recipient nude mice revealed that MPP3 cells were the most effective subset (relative to CLPs). Although several lymphoid genes are expressed by MPP3 cells and Flt3(-) CLPs, the latter only give rise to B cells in the spleen, and Notch1 expression level is not modulated in the blood, as for MPP3 cells. We conclude that CLPs have reached the point where they cannot be a Notch1 target, a limiting condition on the path to T cell engagement., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Thymus transplants can correct deficiencies of the thymus epithelium caused by the complete DiGeorge syndrome or FOXN1 mutations. However, thymus transplants were never used to correct T cell-intrinsic deficiencies because it is generally believed that thymocytes have short intrinsic lifespans. This notion is based on thymus transplantation experiments where it was shown that thymus-resident cells were rapidly replaced by progenitors originating in the bone marrow. In contrast, here we show that neonatal thymi transplanted into interleukin 7 receptor-deficient hosts harbor populations with extensive capacity to self-renew, and maintain continuous thymocyte generation and export. These thymus transplants reconstitute the full diversity of peripheral T cell repertoires one month after surgery, which is the earliest time point studied. Moreover, transplantation experiments performed across major histocompatibility barriers show that allogeneic transplanted thymi are not rejected, and allogeneic cells do not induce graft-versus-host disease; transplants induced partial or total protection to infection. These results challenge the current dogma that thymocytes cannot self-renew, and indicate a potential use of neonatal thymus transplants to correct T cell-intrinsic deficiencies. Finally, as found with mature T cells, they show that thymocyte survival is determined by the competition between incoming progenitors and resident cells.

Common lymphoid progenitors (CLP) are generated in adult bone marrow (BM), but the intermediate steps leading to T cell commitment are unknown, and so is the site at which this commitment occurs. Here, we show that colonies arising in the spleen 12 days after BM injection harbor T cell precursors that are undetectable in BM. These precursors did not generate myeloid cells in vivo but repopulated the thymus and the peripheral T cell compartment much faster than did CLP. Two lineage negative (Lin(-)) subpopulations were distinguished, namely CD44(+) Thy1(-) cells still capable of natural killer generation and transient low-level B cell generation, and T cell-restricted CD44(-) Thy1(+) cells. At a molecular level, frequency of CD3epsilon and preTalpha mRNA was very different in each subset. Furthermore, only the CD44(-) Thy1(+) subset have initiated rearrangements in the T cell receptor beta locus. Thus, this study identifies extramedullary T cell progenitors and will allow easy approach to T cell commitment studies.

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.

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.