Isaac Engel

E2A proteins enforce a proliferation checkpoint in developing thymocytes

E2A proteins regulate multiple stages of thymocyte development and suppress T‐cell lymphoma. The activity of E2A proteins throughout thymocyte development is modulated by signals emanating from the pre‐TCR and TCR. Here we demonstrate that E2A is required for the complete arrest in both differentiation and proliferation observed in thymocytes with defects in proteins that mediate pre‐TCR signaling, including LAT, Lck and Fyn. We show that E2A proteins are required to prevent the accumulation of TCRβ negative cells beyond the pre‐TCR checkpoint. E2A‐deficient thymocytes also exhibit abnormal cell‐cycle progression prior to pre‐TCR expression. Furthermore, we demonstrate that E47 can act in concert with Bcl‐2 to induce cell‐cycle arrest in vitro. These observations indicate that E2A proteins function during early thymocyte development to block cell‐cycle progression prior to the expression of TCRβ. In addition, these data provide further insight into how deficiencies in E2A lead to T lymphoma.

Disruption of pre-TCR expression accelerates lymphomagenesis in E2A-deficient mice

The helix–loop–helix proteins E47 and E12, which are encoded by the E2A gene, regulate several stages of T cell development. In addition, mice deficient for E2A are highly susceptible to thymic lymphoma. Here we report that the development of lymphoma in E2A-deficient mice did not require pre- and recombinase-activating gene expression. Rather, we found that, whereas illegitimate DNA rearrangement did not play a major role in the development of these lymphomas, defects that prevented pre-T cell antigen receptor expression tended to accelerate lymphomagenesis in E2A-deficient mice. These data and previous observations also provide insight into the role of Notch in lymphoma development. Specifically, we propose that Notch activation indirectly modulates E2A activity through induction of pre-Tα expression, ultimately leading to the development of lymphoma.

Transcriptional regulator Id2 controls survival of hepatic NKT cells

Natural killer T cells expressing an invariant T-cell receptor (iNKT) regulate activation of both innate and adaptive immunity in many contexts. iNKT cells accumulate in the liver and rapidly produce prodigious amounts of numerous cytokines upon activation, impacting the immune response to viral infection, immunosurveillance for malignant cells, and liver regeneration. However, little is known about the factors controlling iNKT homeostasis, survival and hepatic localization. Here, we report that the absence of the transcriptional regulator Id2 resulted in a severe, intrinsic defect in the accumulation of hepatic iNKT cells. Id2-deficient iNKT cells showed increased cell death in the liver, although migration and functional activity were not impaired in comparison to Id2-expressing iNKT cells. Id2-deficient iNKT cells exhibited diminished expression of CXCR6, a critical determinant of iNKT cell accumulation in the liver, and of the anti-apoptotic molecules bcl-2 and bcl-XL, compared to Id2-sufficient iNKT cells. Furthermore, survival and accumulation of iNKT cells lacking Id2 expression was rescued by deficiency in bim, a key pro-apoptotic molecule. Thus, Id2 was necessary to establish a hepatic iNKT cell population, defining a role for Id2 and implicating the Id targets, E protein transcription factors, in the regulation of iNKT cell homeostasis.

Loss of T Cell and B Cell Quiescence Precedes the Onset of Microbial Flora-Dependent Wasting Disease and Intestinal Inflammation in Gimap5-Deficient Mice

Homeostatic control of the immune system involves mechanisms that ensure the self-tolerance, survival and quiescence of hematopoietic-derived cells. In this study, we demonstrate that the GTPase of immunity associated protein (Gimap)5 regulates these processes in lymphocytes and hematopoietic progenitor cells. As a consequence of a recessive N-ethyl-N-nitrosourea–induced germline mutation in the P-loop of Gimap5, lymphopenia, hepatic extramedullary hematopoiesis, weight loss, and intestinal inflammation occur in homozygous mutant mice. Irradiated fetal liver chimeric mice reconstituted with Gimap5-deficient cells lose weight and become lymphopenic, demonstrating a hematopoietic cell-intrinsic function for Gimap5. Although Gimap5-deficient CD4+ T cells and B cells appear to undergo normal development, they fail to proliferate upon Ag-receptor stimulation although NF-κB, MAP kinase and Akt activation occur normally. In addition, in Gimap5-deficient mice, CD4+ T cells adopt a CD44highCD62LlowCD69low phenotype and show reduced IL-7rα expression, and T-dependent and T-independent B cell responses are abrogated. Thus, Gimap5-deficiency affects a noncanonical signaling pathway required for Ag-receptor–induced proliferation and lymphocyte quiescence. Antibiotic-treatment or the adoptive transfer of Rag-sufficient splenocytes ameliorates intestinal inflammation and weight loss, suggesting that immune responses triggered by microbial flora causes the morbidity in Gimap5-deficient mice. These data establish Gimap5 as a key regulator of hematopoietic integrity and lymphocyte homeostasis.

The transcription factor Th-POK negatively regulates Th17 differentiation in Vα14i NKT cells.

The majority of mouse Vα14 invariant natural killer T (Vα14i NKT) cells produce several cytokines, including IFNγ and IL-4, very rapidly after activation. A subset of these cells, known as NKT17 cells, however, differentiates in the thymus to preferentially produce IL-17. Here, we show that the transcription factor-known as T helper, Poxviruses, and Zinc-finger and Krüppel family, (Th-POK)-represses the formation of NKT17 cells. Vα14i NKT cells from Th-POK-mutant helper deficient (hd/hd) mice have increased transcripts of genes normally expressed by Th17 and NKT17 cells, and even heterozygosity for this mutation leads to dramatically increased numbers of Vα14i NKT cells that are poised to express IL-17, especially in the thymus and lymph nodes. In addition, using gene reporter mice, we demonstrate that NKT17 cells from wild-type mice express lower amounts of Th-POK than the majority population of Vα14i NKT cells. We also show that retroviral transduction of Th-POK represses the expression of the Th17 master regulator RORγT in Vα14i NKT-cell lines. Our data suggest that NKT17-cell differentiation is intrinsically regulated by Th-POK activity, with only low levels of Th-POK permissive for the differentiation of NKT17 cells.

Co-receptor choice by Vα14i NKT cells is driven by Th-POK expression rather than avoidance of CD8-mediated negative selection

Mouse natural killer T (NKT) cells with an invariant Vα14-Jα18 rearrangement (Vα14 invariant [Vα14i] NKT cells) are either CD4+CD8− or CD4−CD8−. Because transgenic mice with forced CD8 expression in all T cells exhibited a profound NKT cell deficit, the absence of CD8 has been attributed to negative selection. We now present evidence that CD8 does not serve as a coreceptor for CD1d recognition and that the defect in development in CD8 transgene homozygous mice is the result of a reduction in secondary T cell receptor α rearrangements. Thymocytes from mice hemizygous for the CD8 transgene have a less severe rearrangement defect and have functional CD8+ Vα14i NKT cells. Furthermore, we demonstrate that the transcription factor Th, Poxviruses and Zinc finger, and Krüppel family (Th-POK) is expressed by Vα14i NKT cells throughout their differentiation and is necessary both to silence CD8 expression and for the functional maturity of Vα14i NKT cells. We therefore suggest that Th-POK expression is required for the normal development of Vα14i NKT cells and that the absence of CD8 expression by these cells is a by-product of such expression, as opposed to the result of negative selection of CD8-expressing Vα14i NKT cells.

Making memory at birth: understanding the differentiation of natural killer T cells

Glycolipid reactive natural killer T cells with an invariant TCR α-chain (iNKT cells) are a conserved population of T lymphocytes with a distinct anatomical distribution and functional properties. The differentiation pathway of iNKT cells branches off from mainstream thymocyte differentiation at the double positive stage, and recent work has revealed how signaling events early in the iNKT cell pathway imprint a memory-like behavior on these cells. Additionally, unique molecular interactions governing iNKT cell development and tissue distribution have been uncovered recently, building up our knowledge of the complex network of interactions that form this population. Novel autologous antigens for these cells have been identified, although it has not yet been resolved if there is single endogenous antigen responsible for both positive selection and/or peripheral activation.

Neutrophilic Granulocytes Modulate Invariant NKT Cell Function in Mice and Humans

Invariant NKT (iNKT) cells are a conserved αβTCR+ T cell population that can swiftly produce large amounts of cytokines, thereby activating other leukocytes, including neutrophilic granulocytes (neutrophils). In this study, we investigated the reverse relationship, showing that high neutrophil concentrations suppress the iNKT cell response in mice and humans. Peripheral Vα14 iNKT cells from spontaneously neutrophilic mice produced reduced cytokines in response to the model iNKT cell Ag α-galactosyl ceramide and expressed lower amounts of the T-box transcription factor 21 and GATA3 transcription factor than did wild-type controls. This influence was extrinsic, as iNKT cell transcription factor expression in mixed chimeric mice depended on neutrophil count, not iNKT cell genotype. Transcription factor expression was also decreased in primary iNKT cells from the neutrophil-rich bone marrow compared with spleen in wild-type mice. In vitro, the function of both mouse and human iNKT cells was inhibited by coincubation with neutrophils. This required cell–cell contact with live neutrophils. Neutrophilic inflammation in experimental peritonitis in mice decreased iNKT cell T-box transcription factor 21 and GATA3 expression and α-galactosyl ceramide-induced cytokine production in vivo. This was reverted by blockade of neutrophil mobilization. Similarly, iNKT cells from the human peritoneal cavity expressed lower transcription factor levels during neutrophilic peritonitis. Our data reveal a novel regulatory axis whereby neutrophils reduce iNKT cell responses, which may be important in shaping the extent of inflammation.

Autoimmunity-Associated LYP-W620 Does Not Impair Thymic Negative Selection of Autoreactive T Cells

A C1858T (R620W) variation in the PTPN22 gene encoding the tyrosine phosphatase LYP is a major risk factor for human autoimmunity. LYP is a known negative regulator of signaling through the T cell receptor (TCR), and murine Ptpn22 plays a role in thymic selection. However, the mechanism of action of the R620W variant in autoimmunity remains unclear. One model holds that LYP-W620 is a gain-of-function phosphatase that causes alterations in thymic negative selection and/or thymic output of regulatory T cells (Treg) through inhibition of thymic TCR signaling. To test this model, we generated mice in which the human LYP-W620 variant or its phosphatase-inactive mutant are expressed in developing thymocytes under control of the proximal Lck promoter. We found that LYP-W620 expression results in diminished thymocyte TCR signaling, thus modeling a “gain-of-function” of LYP at the signaling level. However, LYP-W620 transgenic mice display no alterations of thymic negative selection and no anomalies in thymic output of CD4+Foxp3+ Treg were detected in these mice. Lck promoter-directed expression of the human transgene also causes no alteration in thymic repertoire or increase in disease severity in a model of rheumatoid arthritis, which depends on skewed thymic selection of CD4+ T cells. Our data suggest that a gain-of-function of LYP is unlikely to increase risk of autoimmunity through alterations of thymic selection and that LYP likely acts in the periphery perhaps selectively in regulatory T cells or in another cell type to increase risk of autoimmunity.

Jarid2 is induced by TCR signalling and controls iNKT cell maturation

Jarid2 is a reported component of three lysine methyltransferase complexes, polycomb repressive complex 2 (PRC2) that methylates histone 3 lysine 27 (H3K27), and GLP-G9a and SETDB1 complexes that methylate H3K9. Here we show that Jarid2 is upregulated upon TCR stimulation and during positive selection in the thymus. Mice lacking Jarid2 in T cells display an increase in the frequency of IL-4-producing promyelocytic leukemia zinc finger (PLZF)(hi) immature invariant natural killer T (iNKT) cells and innate-like CD8(+) cells; Itk-deficient mice, which have a similar increase of innate-like CD8(+) cells, show blunted upregulation of Jarid2 during positive selection. Jarid2 binds to the Zbtb16 locus, which encodes PLZF, and thymocytes lacking Jarid2 show increased PLZF and decreased H3K9me3 levels. Jarid2-deficient iNKT cells perturb Th17 differentiation, leading to reduced Th17-driven autoimmune pathology. Our results establish Jarid2 as a novel player in iNKT cell maturation that regulates PLZF expression by modulating H3K9 methylation.