Antiapoptotic Mcl-1 is critical for the survival and niche-filling capacity of Foxp3⁺ regulatory T cells.

Foxp3⁺ regulatory T (Treg) cells are a crucial immunosuppressive population of CD4⁺ T cells, yet the homeostatic processes and survival programs that maintain the Treg cell pool are poorly understood. Here we report that peripheral Treg cells markedly alter their proliferative and apoptotic rates to rapidly restore numerical deficit through an interleukin 2-dependent and costimulation-dependent process. By contrast, excess Treg cells are removed by attrition, dependent on the Bim-initiated Bak- and Bax-dependent intrinsic apoptotic pathway. The antiapoptotic proteins Bcl-xL and Bcl-2 were dispensable for survival of Treg cells, whereas Mcl-1 was critical for survival of Treg cells, and the loss of this antiapoptotic protein caused fatal autoimmunity. Together, these data define the active processes by which Treg cells maintain homeostasis via critical survival pathways.

Genomic structure and genetic drift in C57BL/6 congenic metabolic mutant mice.

We used a genome-wide single nucleotide polymorphism (SNP) approach to characterize the genomic structures of four representative C57BL/6 (B6) congenic mutant mouse lines to include the A) long-chain acyl-CoA dehydrogenase (Acadl), B) melanocortin 3 receptor (Mc3r), C) endothelial nitric oxide synthase (Nos3), and D) a replacement of mouse apolipoprotein E (Apoe) by human apolipoprotein E-2 (APOE2). We wanted to evaluate the size and flanking genes of the 129 strain origin mutant allele intervals on the B6 background. Additionally, we wanted to evaluate genetic drift among not only the four mutant lines and their respective B6 origin substrains, but also the drift between two commonly used B6 lines obtained from Jackson Laboratory and Taconic. Overall, we found a range of 129 origin interval sizes in the congenic mutant lines analyzed that ranged from a ~2.8 kb human sequence for APOE2 embedded in a 129S6 interval to the largest being a ~16 Mb fragment containing the targeted Nos3 (eNos) gene. Given the range of 129 strain interval sizes, we found 129 strain flanking genes via annotation in genome data bases ranging from one gene both upstream and downstream of the APOE2 allele to seven genes-upstream and five genes-downstream at the Nos3 locus. Furthermore, we found fourteen SNP differences between the Jackson Laboratory and Taconic B6 mice. These genetic differences were associated with marked adiposity differences between the two B6 substrains. This study demonstrates both the fidelity and the caveats of using congenic gene targeted mouse models and recognizing the importance of selecting the appropriately matched wild-type control mouse line.

Acadl-SNP based genotyping assay for long-chain acyl-CoA dehydrogenase deficient mice.

The long-chain acyl-CoA dehydrogenase (LCAD) (Acadl=gene; LCAD=protein) deficient mouse model has been important in evaluating the role of mitochondrial fatty acid oxidation of long-chain fatty acids in metabolic disorders. The insertion vector-based gene targeting strategy used to generate this model has made it difficult to distinguish homozygous and heterozygous genotypes containing targeted Acadl alleles in LCAD-deficient mice. Herein, we describe the design and validation of Acadl SNP genotyping methods capable of distinguishing between heterozygous and homozygous LCAD-deficient mice. The Acadl SNP genotyping assays are effective at allelic discrimination of both C57BL/6 and 129 mouse strain-based Acadl alleles under conditions including, both low purity and quantity genomic DNA templates. This makes the method practical and provides the necessary tools for genotyping the LCAD-deficient mouse model.

Differential IL-2 expression defines developmental fates of follicular versus nonfollicular helper T cells.

In response to infection, naïve CD4+ T cells differentiate into two subpopulations: T follicular helper (TFH) cells, which support B cell antibody production, and non-TFH cells, which enhance innate immune cell functions. Interleukin-2 (IL-2), the major cytokine produced by naïve T cells, plays an important role in the developmental divergence of these populations. However, the relationship between IL-2 production and fate determination remains unclear. Using reporter mice, we found that differential production of IL-2 by naïve CD4+ T cells defined precursors fated for different immune functions. IL-2 producers, which were fated to become TFH cells, delivered IL-2 to nonproducers destined to become non-TFH cells. Because IL-2 production was limited to cells receiving the strongest T cell receptor (TCR) signals, a direct link between TCR-signal strength, IL-2 production, and T cell fate determination has been established.

IL-2 coordinates IL-2-producing and regulatory T cell interplay.

Many species of bacteria use quorum sensing to sense the amount of secreted metabolites and to adapt their growth according to their population density. We asked whether similar mechanisms would operate in lymphocyte homeostasis. We investigated the regulation of the size of interleukin-2 (IL-2)-producing CD4(+) T cell (IL-2p) pool using different IL-2 reporter mice. We found that in the absence of either IL-2 or regulatory CD4(+) T (T reg) cells, the number of IL-2p cells increases. Administration of IL-2 decreases the number of cells of the IL-2p cell subset and, pertinently, abrogates their ability to produce IL-2 upon in vivo cognate stimulation, while increasing T reg cell numbers. We propose that control of the IL-2p cell numbers occurs via a quorum sensing-like feedback loop where the produced IL-2 is sensed by both the activated CD4(+) T cell pool and by T reg cells, which reciprocally regulate cells of the IL-2p cell subset. In conclusion, IL-2 acts as a self-regulatory circuit integrating the homeostasis of activated and T reg cells as CD4(+) T cells restrain their growth by monitoring IL-2 levels, thereby preventing uncontrolled responses and autoimmunity.

A distal conserved sequence element controls Ifng gene expression by T cells and NK cells.

Chromatin dynamics that regulate Ifng gene expression are incompletely understood. By using cross-species comparative sequence analyses, we have identified conserved noncoding sequences (CNSs) upstream of the Ifng gene, one of which, located -22 kb from the transcriptional start site, contains clustered consensus binding sequences of transcription factors that function in T cell differentiation. CNS-22 was uniquely associated with histone modifications typical of accessible chromatin in both T helper 1 (Th1) and Th2 cells and demonstrated significant and selective T-bet (T-box transcription factor expressed in T cells, Tbx21)-dependent binding and enhancer activity in Th1 cells. Deletion of CNS-22 in the context of an Ifng reporter transgene ablated T cell receptor-dependent and -independent Ifng expression in Th1 effectors and similarly blocked expression by cytotoxic T lymphocytes and natural killer cells. Thus, a single distal element may be essential for Ifng gene expression by both innate and adaptive immune effector cell lineages.