Lineage divergence at the first TCR-dependent checkpoint: preferential γδ and impaired αβ T cell development in nonobese diabetic mice

The first TCR-dependent checkpoint in the thymus determines αβ versus γδ T lineage fate and sets the stage for later T cell differentiation decisions. We had previously shown that early T cells in NOD mice that are unable to rearrange a TCR exhibit a defect in checkpoint enforcement at this stage. To determine if T cell progenitors from wild-type NOD mice also exhibit cell-autonomous defects in development, we investigated their differentiation in the Notch-ligand-presenting OP9-DL1 coculture system, as well as by analysis of T cell development in vivo. Cultured CD4 and CD8 double-negative cells from NOD mice exhibited major defects in the generation of CD4 and CD8 double-positive αβ T cells, whereas γδ T cell development from bipotent precursors was enhanced. Limiting dilution and single-cell experiments show that the divergent effects on αβ and γδ T cell development did not spring from biased lineage choice but from increased proliferation of γδ T cells and impaired accumulation of αβ T lineage double-positive cells. In vivo, NOD early T cell subsets in the thymus also show characteristics indicative of defective β-selection, and peripheral αβ T cells are poorly established in mixed bone marrow chimeras, contrasting with strong γδ T as well as B cell repopulation. Thus, NOD T cell precursors reveal divergent, lineage-specific differentiation abnormalities in vitro and in vivo from the first TCR-dependent developmental choice point, which may have consequences for subsequent lineage decisions and effector functions.     

Close link between development and function of gamma-delta T cells

Murine γδ T cells develop as the first T-cell lineage within the fetal thymus and disproportionately localize in mucosal tissues such as lung, skin, uterus, and intestine of adult mice. These unique developmental features and distribution patterns of γδ T cells enable rapid functioning against various insults from pathogens. γδ T cells are also able to respond to local inflammation and consequently regulate the pathogenesis of autoimmune disorders and development of tumors in mice and humans. Hence, it is clinically important to understand the mechanisms that regulate γδ T cell functions. Recent evidence has shown that generations of effector γδ T cell subsets producing IFN-γ, IL-4, and IL-17 are programmed in the murine thymus before their migration to peripheral tissues. This review outlines our current understanding of the development and function of γδ T cells as they influence both innate and acquired immunity.     

Cutting edge: Transitional T3 B cells do not give rise to mature B cells, have undergone selection, and are reduced in murine lupus

As the immediate precursors to mature follicular B cells in splenic development, immature transitional cells are an essential component for understanding late B cell differentiation. It has been shown that T2 cells can give rise to mature B cells; however, whether T3 B cells represent a normal stage of B cell development, which has been widely assumed, has not been fully resolved. In this study, we demonstrate both in vitro and in vivo that T3 B cells do not give rise to mature B cells and are instead selected away from the T1-->T2-->mature B cell developmental pathway and are hyporesponsive to stimulation through the BCR. Significantly reduced numbers of T3 B cells in young lupus-prone mice further suggest that the specificity of this subset holds clues to understanding autoimmunity.     

Prolonged expression of high molecular mass CD45RA isoform during the differentiation of human progenitor thymocytes to CD3+ cells in vitro.

CD45, the leukocyte common Ag, has been shown to characterize T cell development both within the thymus and among peripheral T cells. The work reported here demonstrates that human multinegative (MN) thymocytes, depleted of cells bearing CD3, CD4, CD8, and CD19, express predominantly the high molecular mass CD45RA isoform, and lack low molecular mass CD45RB isoforms and CD45R0 as detected by immunofluorescence. By immunoprecipitation of surface-labeled CD45 molecules from MN thymocytes, a proportion of the CD45 is in fact of low molecular mass but does not include epitopes recognized by CD45R0, nor by CD45RB mAb specific for the p190. This suggests either glycosylation variants of CD45RB/CD45R0 undetectable by our mAb, or underglycosylated CD45RA. MN thymocytes lack TCR-alpha beta mRNA confirming their early developmental stage. Upon culture with IL-2 or with mitogenic combinations of anti-CD2/CD28 mAb, MN thymocytes differentiate to acquire CD3, TCR-alpha beta, and in some cases CD4 and/or CD8. We have predicted that maintenance of CD45RA and lack of CD45R0 expression is fundamental to generative thymic development. If correct, this demands that unlike peripheral T cells, differentiation of MN thymocytes should be accompanied by prolonged expression of high molecular mass CD45 isoforms. Analysis of CD45 isoform expression during MN thymocyte development confirms this prediction and indicates that expression of CD45RA is maintained, at increasing density, for at least 8 to 12 days of culture. Unlike peripheral blood T cells, this is accompanied by the gradual acquisition of firstly the p190 isoforms of CD45RB and later by CD45R0, resulting in a population of CD3+TCR-alpha beta cells coexpressing CD45RA/RBp190/R0. Dot blot analysis of mRNA from differentiating MN thymocytes indicates prolonged expression of mRNA encoding CD45 exons a, b, and c, again in contrast to peripheral T cells which lose all mRNA for alternatively spliced CD45 exons within the first 24 h poststimulation. This is discussed in the context of negative selection during thymic development and interconversion of T cell subsets.     

New insights into the roles of Stat5a/b and Stat3 in T cell development and differentiation

T cell development and differentiation is carefully orchestrated by a series of cytokines. The importance of STAT family proteins in mediating signals by these cytokines is well-known, but new information on the role of STATs in novel aspects of T cell function and T cell subsets continues to accumulate. Recent studies have placed Stat5a/b and Stat3 center stage in T cell development and differentiation. Stat5a/b are indispensable in T regulatory (Treg) cell development and maintenance, and negatively regulate T helper 17 (Th17) cell differentiation. Conversely, Stat3 is essential for Th17 differentiation and inhibits Treg cells. The balance of Treg and Th17 cells is thought to be critical in maintaining immune tolerance, while preserving effective host defense. Therefore, Stat5a/b and Stat3 are emerging to be key players in T cell differentiation and homeostasis.     

The origin of mesoderm in phoronids

Integrin alphaIIb is a cell adhesion molecule expressed in association with beta3 by cells of the megakaryocytic lineage, from committed progenitors to platelets. While it is clear that lymphohemopoietic cells differentiating along other lineages do not express this molecule, it has been questioned whether mammalian hemopoietic stem cells (HSC) and various progenitor cells express it. In this study, we detected alphaIIb expression in midgestation embryo in sites of HSC generation, such as the yolk sac blood islands and the hemopoietic clusters lining the walls of the major arteries, and in sites of HSC migration, such as the fetal liver. Since c-Kit, which plays an essential role in the early stages of hemopoiesis, is expressed by HSC, we studied the expression of the alphaIIb antigen in the c-Kit-positive population from fetal liver and adult bone marrow differentiating in vitro and in vivo into erythromyeloid and lymphocyte lineages. Erythroid and myeloid progenitor activities were found in vitro in the c-Kit(+)alphaIIb(+) cell populations from both origins. On the other hand, a T cell developmental potential has never been considered for c-Kit(+)alphaIIb(+) progenitors, except in the avian model. Using organ cultures of embryonic thymus followed by grafting into athymic nude recipients, we demonstrate herein that populations from murine fetal liver and adult bone marrow contain T lymphocyte progenitors. Migration and maturation of T cells occurred, as shown by the development of both CD4(+)CD8- and CD4-CD8(+) peripheral T cells. Multilineage differentiation, including the B lymphoid lineage, of c-Kit(+)alphaIIb(+) progenitor cells was also shown in vivo in an assay using lethally irradiated congenic recipients. Taken together, these data demonstrate that murine c-Kit(+)alphaIIb(+) progenitor cells have several lineage potentialities since erythroid, myeloid, and lymphoid lineages can be generated.     

Transitional B lymphocyte subsets operate as distinct checkpoints in murine splenic B cell development

Signaling through the Ag receptor is required for peripheral B lymphocyte maturation and maintenance. Defects in components of the B cell receptor (BCR) signalosome result in developmental blocks at the transition from immature (heat-stable Ag (HSA)(high)) to mature (HSA(low)) B cells. Recent studies have subdivided the immature, or transitional, splenic B cells into two subsets, transitional 1 (T1) and transitional 2 (T2) cells. T1 and T2 cells express distinct surface markers and are located in distinct anatomic locations. In this report, we evaluated the BCR signaling capacity of T1 and T2 B cell subsets. In response to BCR engagement, T2 cells rapidly entered cell cycle and resisted cell death. In contrast, T1 cells did not proliferate and instead died after BCR stimulation. Correlating with these results, T2 cells robustly induced expression of the cell cycle regulator cyclin D2 and the antiapoptotic factors A1/Bfl-1 and Bcl-x(L) and exhibited activation of Akt. In contrast, T1 cells failed to up-regulate these markers. BCR stimulation of T2 cells also led to down-regulation of CD21 and CD24 (HSA) expression, resulting in a mature B cell phenotype. In addition, T2 cells from Bruton's tyrosine kinase-deficient Xid mice failed to generate these proliferative and survival responses, suggesting a requirement for the BCR signalosome specifically at the T2 stage. Taken together, these data clearly demonstrate that T2 immature B cells comprise a discrete developmental subset that mediates BCR-dependent proliferative, prosurvival, and differentiation signals. Their distinct BCR-dependent responses suggest unique roles for T1 vs T2 cells in peripheral B cell selection.     

Early mouse embryos produce and release factors with transforming growth factor activity

Summary Previous studies have shown that extracts from mouse embryos at mid and late stages of development contain factors that exhibit transforming growth factor activity. The work reported here demonstrates that cultured mouse embryos at significantly earlier stages of development produce and release factors that exhibit the characteristic property of transforming growth factors. Specifically, the data demonstrate that embryos cultured from the blastocyst stage in serum-containing medium or in serum-free medium release factors that promote the anchorage-independent growth of normal rat kidney fibroblasts. It is shown that these factors are produced and released by cells derived from the inner cell mass and by trophoblasts. The precise developmental stage when production of these factors first begins has not been determined but our findings suggest that these factors are produced by cell types associated with early postimplatation embryos. This work was supported by the Laboratory of Viral Carcinogenesis at the National Cancer Institute and by grants from the National Cancer Institute (CA-36727) and the University of Nebraska Medical Center (22-271-732). Editor's Statement This paper presents evidence that, in an in vitro assay system, early embryonic cells are capable of both synthesizing and secreting TGF-like growth factors, implicating the production of these factors in the events of early development. David W. Barnes     

Phosphodiesterase 7A1 is expressed in human CD4+ naïve T cells at higher levels than in CD4+ memory cells and is not required during their CD3/CD28-dependent activation

PDE7A1 is a cAMP-hydrolyzing phosphodiesterase expressed in lymphoid tissue, where its possible role during T cell activation remains unclear. We have characterized the functional relevance of PDE7A1 in the na?ve (CD4+CD45RA+) and memory (CD4+CD45RO+) subsets of human peripheral CD4+ T cells during CD3/CD28-dependent stimulation. Our results indicate that PDE7A1 is expressed in resting na?ve CD4+ T cells at higher levels than in the corresponding memory cells and that levels of PDE7A1 mRNA are not upregulated upon CD3/CD28 mediated stimulation of these T cell subsets. Treatment with a selective inhibitor of PDE7A1 does not impair CD3/CD28 induced activation of na?ve or memory CD4+ T cells, nor does it increase intracellular cAMP in CD4+ T cells. We conclude that PDE7A1 is not required during CD3/CD28-dependent activation of na?ve and memory CD4+ T cells, but cannot rule out other regulatory roles of PDE7A1 during maturation of CD4+ T cells.     

Variable expression of lineage regulators in differentiated stromal cells indicates distinct mechanisms of differentiation towards common cell fate

Mesenchymal stem cells (MSCs) possess a multi-lineage differentiation capacity that makes them important players in the field of regenerative medicine. MSC populations derived from different tissues or donors have been shown to exhibit variable gene expression patterns. Further, it is widely acknowledged that MSC isolates are heterogeneous mixtures of cells at different developmental stages. However, the heterogeneity of expression of lineage regulators has not been linked to differentiation potential of different MSC populations towards mesenchymal lineages. Here, we analyzed variation of expression of differentiation markers across whole population and between single differentiating cells of multipotent stromal cell populations derived from adipose tissue (AdMSCs) and skin (FBs) of seven donors. The results of the analyses show that all cell populations exhibit similar differentiation potential towards adipocyte, osteoblast and chondrocyte lineages despite tissue type- and donor-specific variations of expression of differentiation-associated genes. Further, we detected variable expression of lineage regulators in individual differentiating cells. Together, our data indicate that single cells of stromal cell populations could use distinct molecular mechanisms to reach a common cell fate.     

The Croonian Lecture, 1992. The key role of the thymus in the body's defence strategies.

For centuries the thymus has remained a mysterious organ with largely unknown functions. The first demonstration of its crucial role in the development of the immune system was reported in 1961, when it was found that mice thymectomized at birth had poorly developed lymphoid tissues, impaired immune reactivities, and an inordinate susceptibility to develop infections. Although thymus lymphocytes were for a long time deemed immunoincompetent, it was shown in 1967 that they could respond to antigen by proliferating to give rise to a progeny of cells which did not secrete antibody (T cells), but which had a remarkable ability to induce bone marrow cells (B cells) to become antibody formers. This was the first unequivocal demonstration of a major division of labour among mammalian lymphocytes. Tremendous progress in our understanding of the function of the thymus and of the T cells derived from it followed. Distinct T cell subsets were characterized and shown to have an essential role in initiating and regulating a variety of immune responses. The ontogenetic events which occurred during their differentiation were mapped, and this allowed studies of the selection of the T cell repertoire. The major histocompatibility complex and associated peptides were shown to govern T cell selection and antigen activation, and the antigen-specific T cell receptor and the genes which code for it were characterized. Future studies should allow some insight into how to activate T cells more effectively for vaccination purposes, and how to switch them off to prevent autoimmune reactions and to induce tolerance to transplanted tissues.     

Strength of stimulus and clonal competition impact the rate of memory CD8 T cell differentiation

The developmental pathways of long-lived memory CD8 T cells and the lineage relationship between memory T cell subsets remain controversial. Although some studies indicate the two major memory T cell subsets, central memory T (T(CM)) and effector memory T (T(EM)), are related lineages, others suggest that these subsets arise and are maintained independently of one another. In this study, we have investigated this issue and examined the differentiation of memory CD8 T cell subsets by tracking the lineage relationships of both endogenous and TCR transgenic CD8 T cell responses after acute infection. Our data indicate that TCR transgenic as well as nontransgenic T(EM) differentiate into T(CM) in the absence of Ag. Moreover, the rate of memory CD8 T cell differentiation from T(EM) into the self-renewing and long-lived pool of T(CM) is influenced by signals received during priming, including Ag levels, clonal competition, and/or the duration of infection. Although some T(EM) appear to not progress to T(CM), the vast majority of T(CM) are derived from T(EM). Thus, long-lasting, Ag-independent CD8 T cell memory results from progressive differentiation of memory CD8 T cells, and the rate of memory T cell differentiation is governed by events occurring early during T cell priming.     

Langerhans cells are required for efficient presentation of topically applied hapten to T cells

Dendritic cells (DC) play a pivotal role in the control of T cell immunity due to their ability to stimulate naive T cells and direct effector function. Murine and human DC are composed of a number of phenotypically, and probably developmentally, distinct subsets, which may play unique roles in the initiation and regulation of T cell responses. The skin is populated by at least two subsets of DC: Langerhans cells (LC), which form a contiguous network throughout the epidermis, and dermal DC. LC have classically been thought vital to initiate T cell responses to cutaneous Ags. However, recent data have highlighted the importance of dermal DC in cutaneous immunity, and the requirement for LC has become unclear. To define the relative roles of LC and dermal DC, we and others generated mouse models in which LC were specifically depleted in vivo. Unexpectedly, these studies yielded conflicting data as to the role of LC in cutaneous contact hypersensitivity (CHS). Extending our initial finding, we demonstrate that topical Ag is inefficiently transported to draining lymph nodes in the absence of LC, resulting in suboptimal priming of T cells and reduced CHS. However, dermal DC may also prime cutaneous T cell responses, suggesting redundancy between the two different skin DC subsets in this model.     

Age-related changes in mature CD4+ T cells: cell cycle analysis

T cell proliferative responses decrease with age, but the mechanisms responsible are unknown. We examined the impact of age on memory and naive CD4(+) T cell entry and progression through the cell cycle using acridine orange to identify cell cycle stage. For both subsets, fewer stimulated cells from old donors were able to enter and progress through the first cell cycle, with an increased number of cells arrested in G(0) and fewer cells in post G(0) phases. The number of dead cells as assessed by sub-G(0) DNA was also significantly greater in the old group. CD4(+) T cells from old mice also exhibited a significant reduction in clonal history as assessed by CFSE staining. This was associated with a significant decline in cyclin D2 mRNA and protein. We propose that decreases in cyclin D2 are at least partially responsible for the proliferative decline found in aged CD4(+) T cells.