Kathryn F. Wildt

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.

The transcription factor Zbtb7b promotes CD4 expression by antagonizing runx-mediated activation of the CD4 silencer

The persistence of CD4 expression is a key event distinguishing the differentiation of MHC class II-restricted thymocytes into CD4 T cells from that of MHC class I-restricted thymocytes into CD8 T cells. The zinc finger transcription factor Zbtb7b (or cKrox or Thpok) is normally expressed in MHC class II-restricted thymocytes and promotes CD4 lineage choice. When expressed in MHC class I-restricted cells, Zbtb7b redirects these cells from their normal CD8 fate to CD4 differentiation, implying that it promotes, directly or not, sustained CD4 expression; the present study has investigated the mechanism of this effect. We demonstrate that, although Zbtb7b does not enhance CD4 expression on its own, it antagonizes the CD4 repression mediated by the transcription factor Runx3, which is normally up-regulated during CD8 differentiation and promotes CD4 silencing. Zbtb7b also antagonizes CD4 repression by the related protein Runx1, which is expressed in CD4 lineage cells. This antagonism is observed both in vitro and in vivo, is transcriptional, and requires domains of Zbtb7b that are essential to its ability to promote CD4 differentiation in vivo. Furthermore, Zbtb7b fails to antagonize Runx in cells treated with histone deacetylase inhibitors, suggesting that Zbtb7b acts by reducing the expression of thus far unknown factors that cooperate with Runx molecules to repress CD4. These findings demonstrate that the transcription factor Zbtb7b promotes CD4 expression by antagonizing Runx-mediated CD4 repression.

The transcription factor Ets1 is important for CD4 repression and Runx3 up-regulation during CD8 T cell differentiation in the thymus

The transcription factor Ets1 contributes to the differentiation of CD8 lineage cells in the thymus, but how it does so is not understood. In this study, we demonstrate that Ets1 is required for the proper termination of CD4 expression during the differentiation of major histocompatability class 1 (MHC I)–restricted thymocytes, but not for other events associated with their positive selection, including the initiation of cytotoxic gene expression, corticomedullary migration, or thymus exit. We further show that Ets1 promotes expression of Runx3, a transcription factor important for CD8 T cell differentiation and the cessation of Cd4 gene expression. Enforced Runx3 expression in Ets1-deficient MHC I–restricted thymocytes largely rescued their impaired Cd4 silencing, indicating that Ets1 is not required for Runx3 function. Finally, we document that Ets1 binds at least two evolutionarily conserved regions within the Runx3 gene in vivo, supporting the possibility that Ets1 directly contributes to Runx3 transcription. These findings identify Ets1 as a key player during CD8 lineage differentiation and indicate that it acts, at least in part, by promoting Runx3 expression.

Heterozygosity for a defective gene for CC chemokine receptor 5 is not the sole determinant for the immunologic and virologic phenotype of HIV- infected long-term nonprogressors

HIV-1-infected long-term nonprogressors are a heterogeneous group of individuals with regard to immunologic and virologic markers of HIV-1 disease. CC chemokine receptor 5 (CCR5) has recently been identified as an important coreceptor for HIV-1 entry into CD4+ T cells. A mutant allele of CCR5 confers a high degree of resistance to HIV-1 infection in homozygous individuals and partial protection against HIV disease progression in heterozygotes. The frequency of CCR5 heterozygotes is increased among HIV-1- infected long-term nonprogressors compared with progressors; however, the host defense mechanisms responsible for nonprogression in CCR5 heterozygotes are unknown. We hypothesized that nonprogressors who were heterozygous for the mutant CCR5 gene might define a subgroup of nonprogressors with higher CD4+ T cell counts and lower viral load compared with CCR5 wild-type nonprogressors. However, in a cohort of 33 HIV-1-infected long-term nonprogressors, those who were heterozygous for the mutant CCR5 gene were indistinguishable from CCR5 wild-type nonprogressors with regard to all measured immunologic and virologic parameters. Although epidemiologic data support a role for the mutant CCR5 allele in the determination of the state of long-term nonprogression in some HIV-1- infected individuals, it is not the only determinant. Furthermore, long-term nonprogressors with the wild-type CCR5 genotype are indistinguishable from heterozygotes from an immunologic and virologic standpoint.

Restricting Zap70 Expression to CD4+CD8+ Thymocytes Reveals a T Cell Receptor–dependent Proofreading Mechanism Controlling the Completion of Positive Selection

Although T cell receptor (TCR) signals are essential for intrathymic T cell–positive selection, it remains controversial whether they only serve to initiate this process, or whether they are required throughout to promote thymocyte differentiation and survival. To address this issue, we have devised a novel approach to interfere with thymocyte TCR signaling in a developmental stage-specific manner in vivo. We have reconstituted mice deficient for Zap70, a tyrosine kinase required for TCR signaling and normally expressed throughout T cell development, with a Zap70 transgene driven by the adenosine deaminase (ADA) gene enhancer, which is active in CD4+CD8+ thymocytes but inactive in CD4+ or CD8+ single-positive (SP) thymocytes. In such mice, termination of Zap70 expression impaired TCR signal transduction and arrested thymocyte development after the initiation, but before the completion, of positive selection. Arrested thymocytes had terminated Rag gene expression and up-regulated TCR and Bcl-2 expression, but failed to differentiate into mature CD4 or CD8 SP thymocytes, to be rescued from death by neglect or to sustain interleukin 7Rα expression. These observations identify a TCR-dependent proofreading mechanism that verifies thymocyte TCR specificity and differentiation choices before the completion of positive selection.

The sequential activity of Gata3 and Thpok is required for the differentiation of CD1d-restricted CD4+ NKT cells.

While most CD4+ T cells are MHC class II‐restricted, a small subset, including the CD1d‐restricted ‘invariant’ NKT (iNKT) cells, are selected on non‐classical MHC‐I or MHC‐I‐like molecules. We previously showed that the sequential activity of two zinc finger transcription factors, Gata3 and Thpok, promotes the differentiation of conventional, MHC II‐restricted thymocytes into CD4+ T cells. In the current study, we show that a Gata3‐Thpok cascade is required for the differentiation of CD4+ iNKT cells. Gata3 is required for iNKT cells to express Thpok, whereas Thpok is needed for proper NKT cell differentiation, and notably for NKT cells to maintain CD4 and terminate CD8 expression. These findings identify the sequential activity of Gata3 and Thpok as a hallmark of CD4+ T‐cell differentiation, regardless of MHC restriction.