Andrea C. Carpenter

Decision checkpoints in the thymus

The development of T cells in the thymus involves several differentiation and proliferation events, during which hematopoietic precursors give rise to T cells ready to respond to antigen stimulation and undergo effector differentiation. This review addresses signaling and transcriptional checkpoints that control the intrathymic journey of T cell precursors. We focus on the divergence of αβ and γδ lineage cells and the elaboration of the αβ T cell repertoire, with special emphasis on the emergence of transcriptional programs that direct lineage decisions.

Formation of Dynamic γ-H2AX Domains along Broken DNA Strands Is Distinctly Regulated by ATM and MDC1 and Dependent upon H2AX Densities in Chromatin

A hallmark of the cellular response to DNA double-strand breaks (DSBs) is histone H2AX phosphorylation in chromatin to generate gamma-H2AX. Here, we demonstrate that gamma-H2AX densities increase transiently along DNA strands as they are broken and repaired in G1 phase cells. The region across which gamma-H2AX forms does not spread as DSBs persist; rather, gamma-H2AX densities equilibrate at distinct levels within a fixed distance from DNA ends. Although both ATM and DNA-PKcs generate gamma-H2AX, only ATM promotes gamma-H2AX formation to maximal distance and maintains gamma-H2AX densities. MDC1 is essential for gamma-H2AX formation at high densities near DSBs, but not for generation of gamma-H2AX over distal sequences. Reduced H2AX levels in chromatin impair the density, but not the distance, of gamma-H2AX formed. Our data suggest that H2AX fuels a gamma-H2AX self-reinforcing mechanism that retains MDC1 and activated ATM in chromatin near DSBs and promotes continued local phosphorylation of H2AX.

Control of the development of CD8[alpha][alpha]+ intestinal intraepithelial lymphocytes by TGF-[beta]

The molecular mechanisms directing the development of TCRαβ+CD8αα+ intestinal intraepithelial lymphocytes (IEL) are not thoroughly understood. Here we show that transforming growth factor-β (TGF-β) controls the development of TCRαβ+CD8αα+ IEL. Mice with either a TGF-β1 null mutation or a T cell-specific deletion of the TGF-β receptor I lacked TCRαβ+CD8αα+ IEL, whereas transgenic mice that over-expressed TGF-β1 had an increased population of TCRαβ+CD8αα+ IEL. Defective development of the TCRαβ+CD8αα+ IEL thymic precursors (CD4-CD8-TCRαβ+CD5+) was observed in the absence of TGF-β. In addition, we showed that TGF-β signaling induced CD8α expression in TCRαβ+CD8αα+ IEL thymic precursors and induced and maintained CD8α expression in peripheral populations of T cells. These data demonstrate a previously unrecognized role for TGF-β in the development of TCRαβ+CD8αα+ IEL and the expression of CD8 in T cells.The molecular mechanisms that direct the development of TCRαβ+CD8αα+ intestinal intraepithelial lymphocytes (IELs) are not thoroughly understood. Here we show that transforming growth factor-β (TGF-β) controls the development of TCRαβ+CD8αα+ IELs. Mice with either a null mutation in the gene encoding TGF-β1 or T cell–specific deletion of TGF-β receptor I lacked TCRαβ+CD8αα+ IELs, whereas mice with transgenic overexpression of TGF-β1 had a larger population of TCRαβ+CD8αα+ IELs. We observed defective development of the TCRαβ+CD8αα+ IEL thymic precursors (CD4−CD8−TCRαβ+CD5+) in the absence of TGF-β. In addition, we found that TGF-β signaling induced CD8α expression in TCRαβ+CD8αα+ IEL thymic precursors and induced and maintained CD8α expression in peripheral populations of T cells. Our data demonstrate a previously unrecognized role for TGF-β in the development of TCRαβ+CD8αα+ IELs and the expression of CD8α in T cells.

Control of the development of CD8αα + intestinal intraepithelial lymphocytes by TGF-β

The molecular mechanisms directing the development of TCRαβ+CD8αα+ intestinal intraepithelial lymphocytes (IEL) are not thoroughly understood. Here we show that transforming growth factor-β (TGF-β) controls the development of TCRαβ+CD8αα+ IEL. Mice with either a TGF-β1 null mutation or a T cell-specific deletion of the TGF-β receptor I lacked TCRαβ+CD8αα+ IEL, whereas transgenic mice that over-expressed TGF-β1 had an increased population of TCRαβ+CD8αα+ IEL. Defective development of the TCRαβ+CD8αα+ IEL thymic precursors (CD4-CD8-TCRαβ+CD5+) was observed in the absence of TGF-β. In addition, we showed that TGF-β signaling induced CD8α expression in TCRαβ+CD8αα+ IEL thymic precursors and induced and maintained CD8α expression in peripheral populations of T cells. These data demonstrate a previously unrecognized role for TGF-β in the development of TCRαβ+CD8αα+ IEL and the expression of CD8 in T cells.The molecular mechanisms that direct the development of TCRαβ+CD8αα+ intestinal intraepithelial lymphocytes (IELs) are not thoroughly understood. Here we show that transforming growth factor-β (TGF-β) controls the development of TCRαβ+CD8αα+ IELs. Mice with either a null mutation in the gene encoding TGF-β1 or T cell–specific deletion of TGF-β receptor I lacked TCRαβ+CD8αα+ IELs, whereas mice with transgenic overexpression of TGF-β1 had a larger population of TCRαβ+CD8αα+ IELs. We observed defective development of the TCRαβ+CD8αα+ IEL thymic precursors (CD4−CD8−TCRαβ+CD5+) in the absence of TGF-β. In addition, we found that TGF-β signaling induced CD8α expression in TCRαβ+CD8αα+ IEL thymic precursors and induced and maintained CD8α expression in peripheral populations of T cells. Our data demonstrate a previously unrecognized role for TGF-β in the development of TCRαβ+CD8αα+ IELs and the expression of CD8α in T cells.

A ThPOK-LRF transcriptional node maintains the integrity and effector potential of post-thymic CD4+ T cells

The transcription factor ThPOK promotes CD4+ T cell differentiation in the thymus. Here, using a mouse strain that allows post-thymic gene deletion, we show that ThPOK maintains CD4+ T lineage integrity and couples effector differentiation to environmental cues after antigenic stimulation. ThPOK preserved the integrity and amplitude of effector responses and was required for proper differentiation of types 1 and 2 helper T cells in vivo by restraining the expression and function of Runx3, a nuclear factor crucial for cytotoxic T cell differentiation. The transcription factor LRF acts redundantly with ThPOK to prevent the transdifferentiation of mature CD4+ T cells into CD8+ T cells. As such, the ThPOK-LRF transcriptional module was essential for CD4+ T cell integrity and responses.

Productive Coupling of Accessible Vβ14 Segments and DJβ Complexes Determines the Frequency of Vβ14 Rearrangement

To elucidate mechanisms that regulate Vβ rearrangement, we generated and analyzed mice with a V(D)J recombination reporter cassette of germline Dβ-Jβ segments inserted into the endogenous Vβ14 locus (Vβ14Rep). As a control, we first generated and analyzed mice with the same Dβ-Jβ cassette targeted into the generally expressed c-myc locus (c-mycRep). Substantial c-mycRep recombination occurred in both T and B cells and initiated concurrently with endogenous Dβ to Jβ rearrangements in thymocytes. In contrast, Vβ14Rep recombination was restricted to T cells and initiated after endogenous Dβ to Jβ rearrangements, but concurrently with endogenous Vβ14 rearrangements. Thus, the local chromatin environment imparts lineage and developmental stage-specific accessibility upon the inserted reporter. Although Vβ14 rearrangements occur on only 5% of endogenous TCRβ alleles, the Vβ14Rep cassette underwent rearrangement on 80–90% of alleles, supporting the suggestion that productive coupling of accessible Vβ14 segments and DJβ complexes influence the frequency of Vβ14 rearrangements. Strikingly, Vβ14Rep recombination also occurs on TCRβ alleles lacking endogenous Vβ to DJβ rearrangements, indicating that Vβ14 accessibility per se is not subject to allelic exclusion.

TCRβ Feedback Signals Inhibit the Coupling of Recombinationally Accessible Vβ14 Segments with DJβ Complexes

Ag receptor allelic exclusion is thought to occur through monoallelic initiation and subsequent feedback inhibition of recombinational accessibility. However, our previous analysis of mice containing a V(D)J recombination reporter inserted into Vβ14 (Vβ14Rep) indicated that Vβ14 chromatin accessibility is biallelic. To determine whether Vβ14 recombinational accessibility is subject to feedback inhibition, we analyzed TCRβ rearrangements in Vβ14Rep mice containing a preassembled in-frame transgenic Vβ8.2Dβ1Jβ1.1 or an endogenous Vβ14Dβ1Jβ1.4 rearrangement on the homologous chromosome. Expression of either preassembled VβDJβC β-chain accelerated thymocyte development because of enhanced cellular selection, demonstrating that the rate-limiting step in early αβ T cell development is the assembly of an in-frame VβDJβ rearrangement. Expression of these preassembled VβDJβ rearrangements inhibited endogenous Vβ14-to-DJβ rearrangements as expected. However, in contrast to results predicted by the accepted model of TCRβ feedback inhibition, we found that expression of these preassembled TCR β-chains did not downregulate recombinational accessibility of Vβ14 chromatin. Our findings suggest that TCRβ-mediated feedback inhibition of Vβ14 rearrangements depends on inherent properties of Vβ14, Dβ, and Jβ recombination signal sequences.

Assembled DJβ Complexes Influence TCRβ Chain Selection and Peripheral Vβ Repertoire

TCRβ chain repertoire of peripheral αβ T cells is generated through the stepwise assembly and subsequent selection of TCRβ V region exons during thymocyte development. To evaluate the influence of a two-step recombination process on Vβ rearrangement and selection, we generated mice with a preassembled Dβ1Jβ1.1 complex on the Jβ1ω allele, an endogenous TCRβ allele that lacks the Dβ2-Jβ2 cluster, creating the Jβ1DJβ allele. As compared with Jβ1ω/ω mice, both Jβ1DJβ/ω and Jβ1DJβ/DJβ mice exhibited grossly normal thymocyte development and TCRβ allelic exclusion. In addition, Vβ rearrangements on Jβ1DJβ and Jβ1ω alleles were similarly regulated by TCRβ-mediated feedback regulation. However, in-frame VβDJβ rearrangements were present at a higher level on the Jβ1DJβ alleles of Jβ1DJβ/ω αβ T cell hybridomas, as compared with on the Jβ1ω alleles. This bias was most likely due to both an increased frequency of Vβ-to-DJβ rearrangements on Jβ1DJβ alleles and a preferential selection of cells with in-frame VβDJβ exons assembled on Jβ1DJβ alleles during the development of Jβ1DJβ/ω αβ T cells. Consistent with the differential selection of in-frame VβDJβ rearrangements on Jβ1DJβ alleles, the Vβ repertoire of αβ T cells was significantly altered during αβ TCR selection in Jβ1DJβ/ω and Jβ1DJβ/DJβ mice, as compared with in Jβ1ω/ω mice. Our data indicate that the diversity of DJβ complexes assembled during thymocyte development influences TCRβ chain selection and peripheral Vβ repertoire.

Posttranscriptional Silencing of VβDJβCβ Genes Contributes to TCRβ Allelic Exclusion in Mammalian Lymphocytes

Feedback inhibition of V(D)J recombination enforces Ag receptor allelic exclusion in mammalian lymphocytes. Yet, in-frame VβDJβ exons can assemble on both alleles in human and mouse αβ T lineage cells. To elucidate mechanisms that enforce TCRβ allelic exclusion in such cells, we analyzed Vβ expression and rearrangement in mice containing a functional Vβ14DJβ1.5Cβ1 gene (Vβ14NT) and/or Vβ8.2DJβ1.1Cβ1 transgene (Vβ8Tg). The majority of Vβ14NT and Vβ8Tg αβ T lineage cells expressed only Vβ14+ or Vβ8+ TCRβ-chains, respectively, and lacked Vβ rearrangements on wild-type TCRβ loci. However, endogenous Vβ rearrangements and αβ T lineage cells expressing endogenous Vβs from wild-type alleles alone or with the prerearranged Vβ in cell surface TCRβ-chains were observed in Vβ14NT and Vβ8Tg mice. Although nearly all Vβ8Tg:Vβ14NT thymocytes and splenic αβ T cells expressed Vβ8+ TCRβ-chains, only half of these lymphocytes expressed Vβ14+ TCRβ-chains, even though similar steady-state levels of Vβ14NT mRNA were expressed in Vβ8+Vβ14+ and Vβ8+Vβ14− populations. Our data demonstrated that posttranscriptional silencing of functionally assembled endogenous VβDJβCβ genes can enforce TCRβ allelic exclusion and reveal another mechanism that contributes to the development of lymphocytes with monospecific Ag receptors.

Control of Regulatory T Cell Differentiation by the Transcription Factors Thpok and LRF

The CD4+ lineage–specific transcription factor Thpok is required for intrathymic CD4+ T cell differentiation and, together with its homolog LRF, supports CD4+ T cell helper effector responses. However, it is not known whether these factors are needed for the regulatory T cell (Treg) arm of MHC class II responses. In this study, by inactivating in mice the genes encoding both factors in differentiated Tregs, we show that Thpok and LRF are redundantly required to maintain the size and functions of the postthymic Treg pool. They support IL-2–mediated gene expression and the functions of the Treg-specific factor Foxp3. Accordingly, Treg-specific disruption of Thpok and Lrf causes a lethal inflammatory syndrome similar to that resulting from Treg deficiency. Unlike in conventional T cells, Thpok and LRF functions in Tregs are not mediated by their repression of the transcription factor Runx3. Additionally, we found that Thpok is needed for the differentiation of thymic Treg precursors, an observation in line with the fact that Foxp3+ Tregs are CD4+ cells. Thus, a common Thpok-LRF node supports both helper and regulatory arms of MHC class II responses.