Ehydel Castro

The Zinc Finger Transcription Factor Zbtb7b Represses CD8-Lineage Gene Expression in Peripheral CD4+ T Cells

How CD4-CD8 differentiation is maintained in mature T cells is largely unknown. The present study has examined the role in this process of the zinc finger protein Zbtb7b, a critical factor for the commitment of MHC II-restricted thymocytes to the CD4+ lineage. We showed that Zbtb7b acted in peripheral CD4+ T cells to suppress CD8-lineage gene expression, including that of CD8 and cytotoxic effector genes perforin and Granzyme B, and was important for the proper repression of interferon-gamma (IFN-gamma) during effector differentiation. The inappropriate expression of IFN-gamma by Zbtb7b-deficient CD4+ T cells required the activities of Eomesodermin and Runx transcription factors. Runx activity was needed for Granzyme B expression, indicating that Runx proteins control expression of the cytotoxic program. We conclude that a key function of Zbtb7b in the mature CD4+ T cell compartment is to repress CD8-lineage gene expression.

Suppression of Dendritic Cell-Derived IL-12 by Endogenous Glucocorticoids Is Protective in LPS-Induced Sepsis

Sepsis, an exaggerated systemic inflammatory response, remains a major medical challenge. Both hyperinflammation and immunosuppression are implicated as causes of morbidity and mortality. Dendritic cell (DC) loss has been observed in septic patients and in experimental sepsis models, but the role of DCs in sepsis, and the mechanisms and significance of DC loss, are poorly understood. Here, we report that mice with selective deletion of the glucocorticoid receptor (GR) in DCs (GRCD11c-cre) were highly susceptible to LPS-induced septic shock, evidenced by elevated inflammatory cytokine production, hypothermia, and mortality. Neutralizing anti-IL-12 antibodies prevented hypothermia and death, demonstrating that endogenous GC-mediated suppression of IL-12 is protective. In LPS-challenged GRCD11c-cre mice, CD8+ DCs were identified as the major source of prolonged IL-12 production, which correlated with elevations of NK cell-derived IFN-γ. In addition, the loss of GR in CD11c+ cells rescued LPS-induced loss of CD8+ DCs but not other DC subsets. Unlike wild-type animals, exposure of GRCD11c-cre mice to low-dose LPS did not induce CD8+ DC loss or tolerance to subsequent challenge with high dose, but neutralization of IL-12 restored the ability of low-dose LPS to tolerize. Therefore, endogenous glucocorticoids blunt LPS-induced inflammation and promote tolerance by suppressing DC IL-12 production.

Histone H3 Lysine 27 demethylases Jmjd3 and Utx are required for T-cell differentiation.

Although histone H3 lysine 27 trimethylation (H3K27Me3) is associated with gene silencing, whether H3K27Me3 demethylation affects transcription and cell differentiation in vivo has remained elusive. To investigate this, we conditionally inactivated the two H3K27Me3 demethylases, Jmjd3 and Utx, in non-dividing intrathymic CD4+ T-cell precursors. Here we show that both enzymes redundantly promote H3K27Me3 removal at, and expression of, a specific subset of genes involved in terminal thymocyte differentiation, especially S1pr1, encoding a sphingosine-phosphate receptor required for thymocyte egress. Thymocyte expression of S1pr1 was not rescued in Jmjd3- and Utx-deficient male mice, which carry the catalytically inactive Utx homolog Uty, supporting the conclusion that it requires H3K27Me3 demethylase activity. These findings demonstrate that Jmjd3 and Utx are required for T-cell development, and point to a requirement for their H3K27Me3 demethylase activity in cell differentiation.

Thpok-independent repression of Runx3 by Gata3 during CD4+ T-cell differentiation in the thymus.

CD4+ helper T cells are essential for immune responses and differentiate in the thymus from CD4+CD8+ “double‐positive” (DP) thymocytes. The transcription factor Runx3 inhibits CD4+ T‐cell differentiation by repressing Cd4 gene expression; accordingly, Runx3 is not expressed in DP thymocytes or developing CD4+ T cells. The transcription factor Thpok is upregulated in CD4‐differentiating thymocytes and required to repress Runx3. However, how Runx3 is controlled at early stages of CD4+ T‐cell differentiation, before the onset of Thpok expression, remains unknown. Here we show that Gata3, a transcription factor preferentially and transiently upregulated by CD4+ T‐cell precursors, represses Runx3 and binds the Runx3 locus in vivo. Accordingly, we show that high‐level Gata3 expression and expression of Runx3 are mutually exclusive. Furthermore, whereas Runx3 represses Cd4, we show that Gata3 promotes Cd4 expression in Thpok‐deficient thymocytes. Thus, in addition to its previously documented role in promoting CD4‐lineage gene‐expression, Gata3 represses CD8‐lineage gene expression. These findings identify Gata3 as a critical pivot of CD4‐CD8 lineage differentiation.

Distinct IL-7 signaling in recent thymic emigrants versus mature naïve T cells controls T-cell homeostasis.

IL‐7 is essential for T‐cell survival but its availability is limited in vivo. Consequently, all peripheral T cells, including recent thymic emigrants (RTEs) are constantly competing for IL‐7 to survive. RTEs are required to replenish TCR diversity and rejuvenate the peripheral T‐cell pool. However, it remains unknown how RTEs successfully compete with resident mature T cells for IL‐7. Moreover, RTEs express low levels of IL‐7 receptors, presumably rendering them even less competitive. Here, we show that, surprisingly, RTEs are more responsive to IL‐7 than mature naïve T cells as demonstrated by markedly increased STAT5 phosphorylation upon IL‐7 stimulation. Nonetheless, adoptive transfer of RTE cells into lymphopenic host mice resulted in slower IL‐7‐induced homeostatic proliferation and diminished expansion compared to naïve donor T cells. Mechanistically, we found that IL‐7 signaling in RTEs preferentially upregulated expression of Bcl‐2, which is anti‐apoptotic but also anti‐proliferative. In contrast, naïve T cells showed diminished Bcl‐2 induction but greater proliferative response to IL‐7. Collectively, these data indicate that IL‐7 responsiveness in RTE is designed to maximize survival at the expense of reduced proliferation, consistent with RTE serving as a subpopulation of T cells rich in diversity but not in frequency.

c-IAP ubiquitin protein ligase activity is required for 4-1BB signaling and CD8+ memory T-cell survival

Cellular inhibitor of apoptosis proteins (c‐IAP) 1 and 2 are widely expressed ubiquitin protein ligases that regulate a variety of cellular functions, including the sensitivity of T cells to costimulation. 4‐1BB is a TNF receptor family member that signals via a complex that includes TRAF family members and the c‐IAPs to upregulate NF‐κB and ERK, and has been implicated in memory T‐cell survival. Here, we show that effector and memory T cells from mice expressing a dominant negative E3‐inactive c‐IAP2 (c‐IAP2H570A) have impaired signaling downstream of 4‐1BB. When infected with lymphocytic choriomeningitis virus, unlike mice in which c‐IAPs were acutely downregulated by c‐IAP antagonists, the primary response of c‐IAP2H570A mice was normal. However, the number of antigen‐specific CD8+ but not CD4+ T cells declined more rapidly and to a greater extent in c‐IAP2H570A mice than in WT controls. Studies with T‐cell adoptive transfer demonstrated that the enhanced decay of memory cells was T‐cell intrinsic. Thus, c‐IAP E3 activity is required for 4‐1BB coreceptor signaling and maintenance of CD8+ T‐cell memory.

Comparing DNA enrichment of proliferating cells following administration of different stable isotopes of heavy water

Deuterated water (2H2O) is a label commonly used for safe quantitative measurement of deuterium enrichment into DNA of proliferating cells. More recently, it has been used for labeling proteins and other biomolecules. Our in vitro - in vivo research reports important stable isotopic labeling enrichment differences into the DNA nucleosides and their isotopologues (e.g. deoxyadenosine (dA) M + 1, dA M + 2, dA M + 3), as well as tumor cell proliferation effects for various forms of commercially available stable heavy water (2H2O, H218O, and 2H218O). Using an in vitro mouse thymus tumor cell line, we determined that H218O provides superior DNA labeling enrichment quantitation, as measured by GC-positive chemical ionization (PCI)-MS/MS. In addition, at higher but physiologically relevant doses, both 2H218O and 2H2O down modulated mouse thymus tumor cell proliferation, whereas H218O water had no observable effects on cell proliferation. The in vivo labeling studies, where normal mouse bone marrow cells (i.e. high turnover) were evaluated post labeling, demonstrated DNA enrichments concordant with measurements from the in vitro studies. Our research also reports a headspace-GC-NCI-MS method, which rapidly and quantitatively measures stable heavy water levels in total body water.

In vivo kinetics and nonradioactive imaging of rapidly proliferating cells in graft-versus-host disease

Hematopoietic stem cell transplantation (HSCT) offers a cure for cancers that are refractory to chemotherapy and radiation. Most HSCT recipients develop chronic graft-versus-host disease (cGVHD), a systemic alloimmune attack on host organs. Diagnosis is based on clinical signs and symptoms, as biopsies are risky. T cells are central to the biology of cGVHD. We found that a low Treg/CD4+ T effector memory (Tem) ratio in circulation, lymphoid, and target organs identified early and established mouse cGVHD. Using deuterated water labeling to measure multicompartment in vivo kinetics of these subsets, we show robust Tem and Treg proliferation in lymphoid and target organs, while Tregs undergo apoptosis in target organs. Since deuterium enrichment into DNA serves as a proxy for cell proliferation, we developed a whole-body clinically relevant deuterium MRI approach to nonradioactively detect cGVHD and potentially allow imaging of other diseases characterized by rapidly proliferating cells.