Coordination of V rearrangements between loci on homologous chromosomes is critical for Ig and TCR allelic exclusion. The Ataxia Telangietasia mutated (ATM) protein kinase promotes DNA repair and activates checkpoints to suppress aberrant Ig and TCR rearrangements. In response to RAG cleavage of Igκ loci, ATM inhibits RAG expression and suppresses further Vκ-to-Jκ rearrangements to enforce Igκ allelic exclusion. Because V recombination between alleles is more strictly regulated for TCRβ and IgH loci, we evaluated the ability of ATM to restrict biallelic expression and V-to-DJ recombination of TCRβ and IgH genes. We detected greater frequencies of lymphocytes with biallelic expression or aberrant V-to-DJ rearrangement of TCRβ or IgH loci in mice lacking ATM. A preassembled DJβ complex that decreases the number of TCRβ rearrangements needed for a productive TCRβ gene further increased frequencies of ATM-deficient cells with biallelic TCRβ expression. IgH and TCRβ proteins drive proliferation of prolymphocytes through cyclin D3 (Ccnd3), which also inhibits VH transcription. We show that inactivation of Ccnd3 leads to increased frequencies of lymphocytes with biallelic expression of IgH or TCRβ genes. We also show that Ccnd3 inactivation cooperates with ATM deficiency to increase the frequencies of cells with biallelic TCRβ or IgH expression while decreasing the frequency of ATM-deficient lymphocytes with aberrant V-to-DJ recombination. Our data demonstrate that core components of the DNA damage response and cell cycle machinery cooperate to help enforce IgH and TCRβ allelic exclusion and indicate that control of V-to-DJ rearrangements between alleles is important to maintain genomic stability.
Copyright © 2014 by The American Association of Immunologists, Inc.

Islet transplantation is a promising treatment for human type 1 diabetes mellitus. Transplantation requires systemic immunosuppression, which has numerous deleterious side effects. Islet antigen-specific regulatory T cells (Tregs) have been shown to protect islet grafts from autoimmune destruction in the nonobese diabetic (NOD) model when co-localized in the kidney capsule. An extra-hepatic transplant site was established by transplanting islet-loaded microporous poly (lactide-co-glycolide) (PLG) scaffolds into abdominal fat. This study examined an autoimmune transplantation model and determined whether co-localized Tregs could protect islet grafts in an extra-hepatic and extra-renal transplant site. Normoglycemia was restored, and co-transplanted Tregs extended graft survival, including several instances of indefinite protection. Transplanted Tregs were replaced by recipient-derived Tregs over time, indicating that islet antigen-specific Tregs induce tolerance to islet grafts through host-derived Tregs. Thus, Tregs provided protection against a diverse repertoire of autoreactive T-cell-receptor specificities mediating diabetes in the NOD model, possibly through a phenomenon previously described as infectious tolerance. Interestingly, the infiltration by Tregs protected a second islet transplant, indicating systemic tolerance to islet antigens. In summary, PLG scaffolds can serve as an alternative delivery system for islet transplantation that allows for the co-localization of immunomodulatory cells within islet grafts and induces long-term graft survival in an autoimmune diabetes model. This method of co-localizing immunomodulatory cells with islets in a clinically translatable transplant site to affect the immune system on a local and systemic level has potential therapeutic implications for human islet transplantation.

The Dicer nuclease generates small RNAs that regulate diverse biological processes through posttranscriptional gene repression and epigenetic silencing of transcription and recombination. Dicer-deficient cells exhibit impaired differentiation, activity, proliferation, and survival. Dicer inactivation in developing mouse lymphocytes impairs their proliferation and survival and alters Ag receptor gene repertoires for largely undefined reasons. To elucidate functions of Dicer in lymphocyte development and Ag receptor locus transcription and recombination, we analyzed mice with conditional Dicer deletion in thymocytes containing unrearranged or prerearranged TCRβ loci. Expression of either a preassembled functional TCRβ gene (Vβ1(NT)) or the prosurvival BCL2 protein inhibited death and partially rescued proliferative expansion of Dicer-deficient thymocytes. Notably, combined expression of Vβ1(NT) and BCL2 completely rescued proliferative expansion of Dicer-deficient thymocytes and revealed that Dicer promotes survival of cells attempting TCRβ recombination. Finally, inclusion of an endogenous preassembled DJβ complex that enhances Vβ recombination increased death and impaired proliferative expansion of Dicer-deficient thymocytes. These data demonstrate a critical role for Dicer in promoting survival of thymocytes experiencing DNA double-strand breaks (DSBs) during TCRβ recombination. Because DSBs are common and ubiquitous in cells, our findings indicate that impaired cellular survival in response to DSBs should be considered when interpreting Dicer-deficient phenotypes.

The parasitic gastrointestinal nematode Nippostrongylus brasiliensis induces massive expansion of T helper type 2 (Th2) cells in the lung and small intestine. Th2 cells are a major source of interleukin-4 and interleukin-13, two cytokines that appear essential for rapid worm expulsion. It is unclear whether all Th2 cells induced during infection are pathogen-specific because Th2 cells might also be induced by parasite-derived superantigens or cytokine-mediated bystander activation. Bystander Th2 polarization could explain the largely unspecific B-cell response during primary infection. Furthermore, it is not known whether protective immunity depends on a polyclonal repertoire of T-cell receptor (TCR) specificities. To address these unresolved issues, we performed adoptive transfer experiments and analysed the TCR-Vβ repertoire before and after infection of mice with the helminth N. brasiliensis. The results demonstrate that all Th2 cells were generated by antigen-specific rather than superantigen-driven or cytokine-driven activation. Furthermore, we show that worm expulsion was impaired in mice with a limited repertoire of TCR specificities, indicating that a polyclonal T-cell response is required for protective immunity.
© 2011 The Authors. Immunology © 2011 Blackwell Publishing Ltd.

The genomic organization of TCRbeta loci enables Vbeta-to-DJbeta2 rearrangements on alleles with assembled VbetaDJbetaCbeta1 genes, which could have deleterious physiologic consequences. To determine whether such Vbeta rearrangements occur and, if so, how they might be regulated, we analyzed mice with TCRbeta alleles containing preassembled functional VbetaDJbetaCbeta1 genes. Vbeta10 segments were transcribed, rearranged, and expressed in thymocytes when located immediately upstream of a Vbeta1DJbetaCbeta1 gene, but not on alleles with a Vbeta14DJbetaCbeta1 gene. Germline Vbeta10 transcription was silenced in mature alphabeta T cells. This allele-dependent and developmental stage-specific silencing of Vbeta10 correlated with increased CpG methylation and decreased histone acetylation over the Vbeta10 promoter and coding region. Transcription, rearrangement, and expression of the Vbeta4 and Vbeta16 segments located upstream of Vbeta10 were silenced on alleles containing either VbetaDJbetaCbeta1 gene; sequences within Vbeta4, Vbeta16, and the Vbeta4/Vbeta16-Vbeta10 intergenic region exhibited constitutive high CpG methylation and low histone acetylation. Collectively, our data indicate that the position of Vbeta segments relative to assembled VbetaDJbetaCbeta1 genes influences their rearrangement and suggest that DNA sequences between Vbeta segments may form boundaries between active and inactive Vbeta chromatin domains upstream of VbetaDJbetaCbeta genes.