Gilbert J. Kersh

Estrogen Induces Thymic Atrophy by Eliminating Early Thymic Progenitors and Inhibiting Proliferation of β-Selected Thymocytes

Although it has been established that high levels of estrogen can induce thymic involution, the mechanism by which this happens is not known. We have found that daily i.p. injections of the synthetic estrogen 17-β-estradiol reduce thymus cellularity by 80% over a period of 4–6 days. Although the atrophy is most strikingly observed in the CD4/CD8 double-positive (DP) thymic subset, the loss of thymocytes is not accompanied by a significant increase in thymocyte apoptosis, suggesting that direct killing of cells may not be the dominant means by which estrogens induce thymic atrophy. Instead, we find that estradiol drastically reduces the lineage-negative, Flt3+Sca-1+c-Kit+ population in the bone marrow, a population that contains thymic homing progenitors. Within the thymus, we observe that estradiol treatment results in a preferential depletion of early thymic progenitors. In addition, we find that estradiol leads to a significant reduction in the proliferation of thymocytes responding to pre-TCR signals. Reduced proliferation of DN3 and DN4 cell subsets is likely the major contributor to the reduction in DP thymocytes that is observed. The reduction in early thymic progenitors is also likely to contribute to thymic atrophy, as we show that estradiol treatment can reduce the size of Rag1-deficient thymuses, which lack pre-TCR signals and DP thymocytes.

Thymocyte Development in Early Growth Response Gene 1-Deficient Mice

Early growth response gene 1 (Egr1) codes for a transcriptional regulator that contains a zinc-finger DNA binding domain. Egr1 expression is induced by a variety of extracellular stimuli including TCR-ligand interactions. Its pattern of expression in the thymus and dependence on ERK activation have led to speculation that it has a role in T cell development, but the exact nature of this role has been undefined. To more clearly define the role of Egr1 in thymocyte development, we have analyzed thymocytes from Egr1-deficient mice. We find that thymuses from Egr1-deficient mice contain twice as many cells as age-matched controls, and the increase in thymocyte number is apparent at the early CD4/CD8 double negative stage of development. Subsequent maturation to the CD4/CD8 double positive stage and survival of the double positive cells both appear normal in Egr1-deficient animals. We also find that Egr1 promotes positive selection of both CD4 and CD8 single positive cells without playing a major role in negative selection. Egr1 influences positive selection by enhancing expression of the helix-loop-helix inhibitor Id3 and the anti-apoptosis molecule bcl-2. Thus, Egr1 translates developmental signals into appropriate changes in gene expression at multiple stages of thymocyte development.

Opposing regulation of T cell function by Egr‐1/NAB2 and Egr‐2/Egr‐3

TCR‐induced NF‐AT activation leads to the up‐regulation of multiple genes involved in T cell anergy. Since NF‐AT is also involved in T cell activation, we have endeavored to dissect TCR‐induced activating and inhibitory genetic programs. This approach revealed roles for the early growth response (Egr) family of transcription factors and the Egr coactivator/corepressor NGFI‐A‐binding protein (NAB)2 in regulating T cell function. TCR‐induced Egr‐1 and NAB2 enhance T cell function, while Egr‐2 and Egr‐3 inhibit T cell function. In this report, we demonstrate that Egr‐2 and Egr‐3 are induced by NF‐AT in the absence of AP‐1, while Egr‐1 and NAB2 both require AP‐1‐mediated transcription. Our data suggest that Egr‐3 is upstream of Egr‐2, and that mechanistically Egr‐2 and Egr‐3 suppress Egr‐1 and NAB2 expression. Functionally, T cells from Egr‐2 and Egr‐3 null mice are hyperresponsive while T cells from Egr‐3 transgenic, overexpressing mice are hyporesponsive. Furthermore, an in vivo model of autoimmune pneumonitis reveals that T cells from Egr‐3 null mice hasten death while Egr‐3‐overexpressing T cells cause less disease. Overall, our data suggest that just as the Egr/NAB network of genes control cell fate in other systems, TCR‐induced Egr‐1, 2, 3 and NAB2 control the fate of antigen recognition in T cells.

Murine pregnancy leads to reduced proliferation of maternal thymocytes and decreased thymic emigration.

During mammalian pregnancy the maternal thymus undergoes significant involution, and then recovers in size after birth. The mechanism behind this involution is not known, but it has been suggested that elevated levels of hormones during pregnancy induce the involution. We have recently shown that injection of 17β‐oestradiol into mice causes loss of early thymocyte precursors and inhibits proliferation of developing thymocytes. This suggests that elevated oestrogen in pregnancy may contribute to thymic involution. We have investigated this idea by examining the fate of thymocytes during mouse pregnancy in much greater detail than has been previously reported. Looking over a broad time–course, we find that pregnancy does not affect thymocyte precursor populations in the bone marrow, but induces a profound loss of early thymic progenitors in the thymus as early as day 12·5 of pregnancy. This loss is accompanied by decreased thymocyte proliferation, which returns to normal 2–4 days postpartum. No enhancement of apoptosis is detectable at any stage of pregnancy. We also find that there is a reduction in recent thymic emigrants after oestrogen treatment and at day 17·5 of pregnancy, suggesting that thymic involution during pregnancy influences the peripheral T‐cell repertoire. The similarities between oestrogen‐mediated involution and pregnancy‐mediated involution suggest that oestrogen is a significant contributor to loss of thymocyte cellularity during pregnancy, and probably functions primarily by reducing thymocyte proliferation.

Early growth response gene 3 regulates thymocyte proliferation during the transition from CD4-CD8- to CD4+CD8+.

In thymocytes developing in the αβ lineage, the transition from CD4, CD8 double negative (DN) to CD4, CD8 double positive (DP) is associated with several rounds of cell division and changes in the expression of multiple genes. This transition is induced by the formation of a pre-TCR that includes a rearranged TCR β-chain and the pre-TCR α-chain. The mechanism by which the pre-TCR influences both gene expression and proliferation has not been defined. We have evaluated the role played by early growth response gene 3 (Egr3) in translating pre-TCR signals into differentiation and proliferation. Egr3 is a transcriptional regulator that contains a zinc-finger DNA binding domain. We find that Egr3-deficient mice have a reduced number of thymocytes compared with wild-type mice, and that this is due to poor proliferation during the DN to DP transition. Treatment of both Egr3+/+ and Egr3−/− mice on the Rag1−/− background with anti-CD3ε Ab in vivo results in similar differentiation events, but reduced cell recovery in the Egr3−/− mice. We have also generated transgenic mice that express high levels of Egr3 constitutively, and when these mice are bred onto a Rag1−/− background they exhibit increased proliferation in the absence of stimulation and have pre-TCR α-chain and CD25 down-regulation, as well as increased Cα expression. The results show that Egr3 is an important regulator of proliferation in response to pre-TCR signals, and that it also may regulate some specific aspects of differentiation.

Sustained Early Growth Response Gene 3 Expression Inhibits the Survival of CD4/CD8 Double-Positive Thymocytes

In the absence of selection, CD4+, CD8+ double-positive (DP) thymocytes will die after 3–4 days. The mechanism for regulating the life span of DP cells is unknown. Previously, we demonstrated that the zinc finger transcription factor, early growth response gene 3 (Egr3), promotes proliferation during the transition from double negative (DN) to DP. In this study we demonstrate a novel role for Egr3 in controlling DP thymocyte survival in mice. Constitutive transgenic expression of Egr3 in thymocytes increases apoptosis among DP cells and shortens their survival in vitro. In addition, DP cells in Egr3 transgenic mice have poor expression of TCRα, and based on the predominant usage of 3′ Vα and 5′ Jα gene segments, the low level of TCRα expression is a result of DP death soon after the initiation of TCRα rearrangements. Constitutive transgenic expression of Egr3 results in poor expression of Bcl-xL and the thymic isoform of retinoic acid receptor-related orphan receptor γ (RORγt) in DP thymocytes, two molecules that are required in DP cells for normal life span. Egr3 expression induced by pre-TCR signals in nontransgenic mice is transient and returns to background levels before RORγt or Bcl-xL is induced. The data support a model in which Egr3 must be transiently induced in response to pre-TCR signals, so that the expression of the prosurvival molecules, RORγt and Bcl-xL, can be elevated only after the proliferative signal provided by Egr3 has subsided.

Early Growth Response-1 Is Required for CD154 Transcription

CD154 (CD40 ligand) expression on CD4 T cells is normally tightly controlled, but abnormal or dysregulated expression of CD154 has been well documented in autoimmune diseases, such as systemic lupus erythematosus. Beyond regulation by NFAT proteins, little is known about the transcriptional activation of the CD154 promoter. We identified a species-conserved purine-rich sequence located adjacent to the CD154 transcriptional promoter proximal NFAT site, which binds early growth response (Egr) transcription factors. Gel shift assays and chromatin immunoprecipitation assays reveal that Egr-1, Egr-3, and NFAT1 present in primary human CD4 T cells are capable of binding this combinatorial site in vitro and in vivo, respectively. Multimerization of this NFAT/Egr sequence in the context of a reporter gene demonstrates this sequence is transcriptionally active upon T cell activation in primary human CD4 T cells. Overexpression of Egr-1, but not Egr-3, is capable of augmenting transcription of this reporter gene as well as that of an intact CD154 promoter. Conversely, overexpression of small interfering RNA specific for Egr-1 in primary human CD4 T cells inhibits CD154 expression. Similarly, upon activation, CD154 message is notably decreased in splenic CD4 T cells from Egr-1-deficient mice compared with wild-type controls. Our data demonstrate that Egr-1 is required for CD154 transcription in primary CD4 T cells. This has implications for selective targeting of Egr family members to control abnormal expression of CD154 in autoimmune diseases such as systemic lupus erythematosus.

Regulation of Bim by TCR Signals in CD4/CD8 Double-Positive Thymocytes

Bim, a BH3-only Bcl-2 family member, is required for apoptosis of thymocytes in response to negative selection signals. Regulation of the apoptotic activity of Bim during negative selection is not understood. In this study we demonstrate that in murine thymocytes undergoing apoptosis in response to anti-CD3ε injection, levels of Bim protein expression do not change. In immature thymocytes, Bim is associated with mitochondria before stimulation and is not regulated by a change in subcellular localization during apoptosis. We also show that BimEL is rapidly phosphorylated in thymocytes in response to CD3ε cross-linking both in vivo and in vitro, and that phosphorylation is sustained for at least 24 h. Analysis of MHC-deficient mice shows that phosphorylation of Bim occurs in CD4/CD8 double-positive thymocytes and does not depend on activation of mature T cells. We also find that TCR cross-linking on thymocytes induces an increase in the proportion of Bcl-xL bound to Bim at late time points. Our results favor a model in which strong TCR signals regulate the apoptotic activity of Bim by phosphorylation and subsequent changes in binding to Bcl-xL in immature thymocytes.

Control of Recent Thymic Emigrant Survival by Positive Selection Signals and Early Growth Response Gene 1

Early growth response gene 1 (Egr1) is a transcriptional regulator whose expression can be induced by multiple signals including the TCR. Egr1 has been shown to promote positive selection, but an investigation of its role in T cell homeostasis has not been reported. The possibility that similar signals control both positive selection and peripheral T cell homeostasis led us to investigate the role of Egr1 in the maintenance of peripheral T cells. We have found that on TCR transgenic backgrounds, Egr1-deficient mice have a reduction in their number of naive T cells. Although Egr1-deficient animals have a low percentage of mature thymocytes due to inefficient positive selection, the absolute number of mature thymocytes is only slightly reduced due to increased thymus size in Egr1-deficient mice. Despite possessing near normal numbers of mature thymocytes, we find that Egr1-deficient mice have poor accumulation of recent thymic emigrants (RTE) in the periphery. The poor accumulation of RTE in Egr1-deficient mice appears to originate from decreased survival of mature thymocytes and RTE, which we have observed both in vitro and in vivo. These findings suggest that an Egr1-mediated signal during positive selection promotes not only the production of single positive thymocytes, but also the survival of selected thymocytes until they can become established in the periphery.

MAP kinase phosphatase activity sets the threshold for thymocyte positive selection

Phosphorylation of MAP kinases is important for proper translation of T cell antigen receptor (TCR) signals into thymocyte cell fates, but the role of MAP kinase phosphatase (MKP) activity in thymocyte development has not been characterized. To explore the role of MKP in thymocytes, we constructed a double mutant MKP-3 (DM-MKP3) that acts as a dominant-negative inhibitor of ERK- and JNK-specific MKP. Thymocytes developing in the presence of DM-MKP3 have enhanced frequencies of both CD4 and CD8 mature, single-positive cells and no increase in apoptosis. Expression of DM-MKP3 also results in an increased proportion of thymocytes with high levels of both CD69 and TCRβ, suggesting that the increased proportion of mature thymocytes is the result of an increased probability that CD4+CD8+ cells will be positively selected. Thus, MKP activity controls thymocyte cell fate by regulating the threshold of TCR signaling that is able to induce positive selection.