Phenotypic plasticity of T cell progenitors upon exposure to Notch ligands

Despite many efforts, the nature of thymic immigrants that give rise to T cells has remained obscure, especially since it became known that extrathymic lineage-negative, Sca-1–positive, c-kit high progenitor cells differ from intrathymic early T cell progenitors (ETPs) by functional potential and dependence on Notch signaling. After our observation that intrathymic T cell precursors expressing a human CD25 reporter under control of pre-TCRα regulatory elements almost exclusively have the ETP phenotype, we have analyzed the phenotypic changes of reporter-expressing common lymphoid progenitor (CLP) cells in the bone marrow when cultured on Delta-like 1–expressing stromal cells. We note that these quickly adopt the phenotype of double negative (DN)2 thymocytes with little display of the ETP phenotype. Our data suggest that common lymphoid progenitor (CLP) cells could be responsible for the rapid reconstitution of thymus function after bone marrow transplantation since CLP cells in the blood have the capacity to rapidly enter the thymus and become DN2 thymocytes

T cell receptor–instructed αβ versus γδ lineage commitment revealed by single-cell analysis

αβ and γδ T cell lineages develop in the thymus from a common precursor. It is unclear at which stage of development commitment to these lineages takes place and in which way T cell receptor signaling contributes to the process. Recently, it was demonstrated that strong TCR signals favor γδ lineage development, whereas weaker TCR signals promote αβ lineage fate. Two models have been proposed to explain these results. The first model suggests that commitment occurs after TCR expression and TCR signaling directly instructs lymphocytes to adopt one or the other lineage fate. The second model suggests that commitment occurs before TCR expression and that TCR signaling merely confirms the lineage choice. By tracing the fate of single T cell precursors, this study shows that there is no commitment to either the αβ or γδ lineage before TCR expression and that modulation of TCR signaling in progeny of a single TCR-expressing cell changes lineage commitment

Differential synergy of Notch and T cell receptor signaling determines αβ versus γδ lineage fate

Thymic precursors expressing the pre–T cell receptor (TCR), the γδTCR, or the αβTCR can all enter the CD4+8+ αβ lineage, albeit with different efficacy. Here it is shown that proliferation and differentiation of precursors with the different TCRs into αβ lineage cells require Notch signaling at the DN3 stage of thymic development. At the DN4 stage, Notch signaling still significantly contributes to the generation of αβ T cells. In particular, in αβ lineage commitment, the pre-TCR synergizes more efficiently with Notch signals than the other two TCRs, whereas γδTCR-expressing cells can survive and expand in the absence of Notch signals, even though Notch signaling enhances their proliferation. These observations suggest a new model of αβ versus γδ lineage choice in which lineage fate is determined by the extent of synergy between TCR and Notch signaling and in which the evolutionarily recent advent of the cell-autonomously signaling pre-TCR increased the efficacy of αβ T cell generation

Severe Developmental B Lymphopoietic Defects in Foxp3-Deficient Mice are Refractory to Adoptive Regulatory T Cell Therapy

The role of Foxp3-expressing regulatory T (Treg) cells in tolerance and autoimmunity is well-established. However, although of considerable clinical interest, the role of Treg cells in the regulation of hematopoietic homeostasis remains poorly understood. Thus, we analysed B and T lymphopoiesis in the scurfy (Sf) mouse model of Treg cell deficiency. In these experiments, the near-complete block of B lymphopoiesis in the BM of adolescent Sf mice was attributed to autoimmune T cells. We could exclude a constitutive lympho-hematopoietic defect or a B cell-intrinsic function of Foxp3. Efficient B cell development in the BM early in ontogeny and pronounced extramedullary B lymphopoietic activity resulted in a peripheral pool of mature B cells in adolescent Sf mice. However, marginal zone B and B-1a cells were absent throughout ontogeny. Developmental B lymphopoietic defects largely correlated with defective thymopoiesis. Importantly, neonatal adoptive Treg cell therapy suppressed exacerbated production of inflammatory cytokines and restored thymopoiesis but was ineffective in recovering defective B lymphopoiesis, probably due to a failure to compensate production of stroma cell-derived IL-7 and CXCL12. Our observations on autoimmune-mediated incapacitation of the BM environment in Foxp3-deficient mice will have direct implications for the rational design of BM transplantation protocols for patients with severe genetic deficiencies in functional Foxp3+ Treg cells

The Eδ enhancer controls the generation of CD4−CD8− αβTCR-expressing T cells that can give rise to different lineages of αβ T cells

It is well established that the pre–T cell receptor for antigen (TCR) is responsible for efficient expansion and differentiation of thymocytes with productive TCRβ rearrangements. However, Ptcra- as well as Tcra-targeting experiments have suggested that the early expression of Tcra in CD4−CD8− cells can partially rescue the development of αβ CD4+CD8+ cells in Ptcra-deficient mice. In this study, we show that the TCR Eδ but not Eα enhancer function is required for the cell surface expression of αβTCR on immature CD4−CD8− T cell precursors, which play a crucial role in promoting αβ T cell development in the absence of pre-TCR. Thus, αβTCR expression by CD4−CD8− thymocytes not only represents a transgenic artifact but occurs under physiological conditions

Induced B Cell Development in Adult Mice

We employed the B-Indu-Rag1 model in which the coding exon of recombination-activating gene 1 (Rag1) is inactivated by inversion. It is flanked by inverted loxP sites. Accordingly, B cell development is stopped at the pro/pre B-I cell precursor stage. A B cell-specific Cre recombinase fused to a mutated estrogen receptor allows the induction of RAG1 function and B cell development by application of Tamoxifen. Since Rag1 function is recovered in a non-self-renewing precursor cell, only single waves of development can be induced. Using this system, we could determine that B cells minimally require 5 days to undergo development from pro/preB-I cells to the large and 6 days to the small preB-II cell stage. First immature transitional (T) 1 and T2 B cells could be detected in the bone marrow at day 6 and day 7, respectively, while their appearance in the spleen took one additional day. We also tested a contribution of adult bone marrow to the pool of B-1 cells. Sublethally irradiated syngeneic WT mice were adoptively transferred with bone marrow of B-Indu-Rag1 mice and B cell development was induced after 6 weeks. A significant portion of donor derived B-1 cells could be detected in such adult mice. Finally, early VH gene usage was tested after induction of B cell development. During the earliest time points the VH genes proximal to D/J were found to be predominantly rearranged. At later time points, the large family of the most distal VH prevailed

(A) Single pTα CD4CD8CD25 TCRγδ cells were sorted onto OP9-DL1 monolayers

On day 7, the contents of each well were divided into 3 parts. One part was analyzed by FACS for surface marker expression (CD45, CD4, CD8, TCRγδ, and TCRβ), the other two parts were transferred to wells with fresh OP9-DL1 monolayers coated with TCRγδ antibodies or uncoated. On day 14 cells were again analyzed for surface marker expression, and cells from anti-TCRγδ–coated wells were further transferred to uncoated wells with fresh OP9DL1 monolayers and analyzed for marker expression on day 19. (B) The proportion of wells containing DN or both DN and DP cells is shown. Numbers represent percentages of particular wells among the total number of wells that contained CD45 cells at any time point. (C) A representative well containing γδ lineage cells only is shown. CD4CD8TCRγδ cells are detected at least at one time point, and CD4CD8 cells are absent at all time points. (D) A representative well containing αβ and γδ lineage cells only is shown. CD4CD8 cells are present before anti-TCRγδ stimulation. Only CD4CD8TCRγδ cells are detected in the presence of anti-TCRγδ antibody and after its removal. Histogram below shows comparison of surface expression of TCRγδ on DN and DP cells in the same well on day 14 of culture without antibody. Numbers in quadrants indicate the percentage of cells. Red numbers above plots refer to absolute cell number.<p><b>Copyright information:</b></p><p>Taken from "T cell receptor–instructed αβ versus γδ lineage commitment revealed by single-cell analysis"</p><p></p><p>The Journal of Experimental Medicine 2008;205(5):1173-1186.</p><p>Published online 12 May 2008</p><p>PMCID:PMC2373848.</p><p></p

Notch1-dependent lymphomagenesis is assisted by but does not essentially require pre-TCR signaling

Overexpression of intracellular Notch plays an important role in the generation of human acute lymphoblastic T cell leukemia (T-ALL). In mouse models, it was shown that Notch-dependent T-ALL required pre-TCR signaling. Here we show that pre-TCR signaling is required to condition mice for Notch-dependent transformation but that it is not required to sustain malignant growth of T-ALL. In contrast to previous studies, we found that disease development does not require pre-TCR but that it can be accelerated in Rag2-/- mice by transient mimicking of pre-TCR signals. (Blood. 2006;108:305-310

(A) Surface expression of CD4 and CD8α by ex vivo pTα thymocytes (left) or by cells derived from TCRγδCD25 pTα thymocytes cultured for 8 d on OP9-DL1 monolayer (right)

(B) CFSE dilution profiles of TCRγδCD25 pTα thymocytes cultured on an OP9-DL1 monolayer for 4 d with (red) or without (blue and green) TCRγδ antibody. Gated on all live cells (blue and red) or on CD4CD8 cells only (green). (C) Surface expression of TCRβ (left), CD8β (middle), and TCRγδ (right) on ex vivo (top) or generated in cell culture (as in A; bottom) pTα DP cells (red). Unstained (for TCRβ and CD8β) thymocytes or thymocytes from Rag2 TCRβ transgenic mice (for TCR γδ) were used as negative control (shaded). TCRγδ expression on DN pTα thymocytes is shown as positive control (dotted). (D, left) Kinetics of expansion and differentiation of TCRγδCD25 pTa in OP9-DL1 co-cultures. (D, right) CD4CD8 cells were sorted from OP9-DL1 co-culture (no antibody) on day 14 and cultured for an additional 7 d. CD4/CD8 expression was analyzed.<p><b>Copyright information:</b></p><p>Taken from "T cell receptor–instructed αβ versus γδ lineage commitment revealed by single-cell analysis"</p><p></p><p>The Journal of Experimental Medicine 2008;205(5):1173-1186.</p><p>Published online 12 May 2008</p><p>PMCID:PMC2373848.</p><p></p

(A) TCRγδ cells derived from spleen (left) or thymus (right) of WT mice were co-cultured on OP9-DL1 monolayers

On day 7 of co-culture CD4 and CD8 expression was analyzed by flow cytometry. (B) Thymocytes from pTα (middle and right) or TCRδ (left) mice were stained with CD4, CD8, TCRγδ, CD25, and CD24 antibodies. TCRγδCD25, TCRγδCD25CD24, and TCRγδCD25CD24 cells were sorted from the thymi of pTα mice. Post-sort reanalysis of TCRγδCD25 cells is shown on the right. The applied gates are shown above each plot. Unstained thymocytes were used as negative control for CD24 staining (shaded). (C) Sorted subsets (as described in B) were co-cultured with OP9-DL1 in wells coated with anti-TCRγδ antibodies (C, bottom row) or in uncoated wells (C, top row). On day 5, cells were transferred to a fresh OP9-DL1 monolayer in uncoated wells. On day 8, cells were harvested and analyzed as in A. (D) 5,000 of the TCRγδCD25 thymocytes were cultured on OP9-DL1 (left) or OP9-GFP (right) monolayers with (bottom) or without (top) anti-TCRγδ coating. On day 4, cells were transferred to a fresh feeder of the same type in uncoated wells. On day 8, cells were harvested and analyzed as in A. Absolute cell numbers are shown in red on the top of each plot. (E) TCRγδCD25 thymocytes were cultured on an OP9-DL1 monolayer, as in D. CD24 expression on TCRγδ DN cells was analyzed on day 8 of culture. Unstained thymocytes were used as negative control for CD24 staining (shaded). Numbers in each gate indicate the percentage of cells.<p><b>Copyright information:</b></p><p>Taken from "T cell receptor–instructed αβ versus γδ lineage commitment revealed by single-cell analysis"</p><p></p><p>The Journal of Experimental Medicine 2008;205(5):1173-1186.</p><p>Published online 12 May 2008</p><p>PMCID:PMC2373848.</p><p></p