Robert M. Samstein

CD4+ Regulatory T Cells Control TH17 Responses in a Stat3-Dependent Manner

Outfoxing Immune Excess Immune responses are kept in check by Foxp3-expressing CD4+-regulatory T cells (Tregs) through a variety of mechanisms. Expression of specific transcription factors directs Treg responses into distinct T helper cell lineages; however, the transcription factors that regulate particular helper lineages have not been completely characterized. Chaudhry et al. (p. 986, published online 1 October) show that the transcription factor Stat3, that is required for the initial differentiation of TH17-effector T cells, is also required for Treg cell-mediated suppression of TH17-mediated immune responses. Mice carrying a Treg cellspecific deletion in Stat3 succumb to an intestinal inflammatory disease driven by uncontrolled TH17 responses. Thus, different classes of immune responses can result from the expression of helper lineage–specific transcription factors. Suppressor T cells regulate different classes of immune responses through induction of specific transcription factors. Distinct classes of protective immunity are guided by activation of STAT transcription factor family members in response to environmental cues. CD4+ regulatory T cells (Tregs) suppress excessive immune responses, and their deficiency results in a lethal, multi-organ autoimmune syndrome characterized by T helper 1 (TH1) and T helper 2 (TH2) CD4+ T cell–dominated lesions. Here we show that pathogenic TH17 responses in mice are also restrained by Tregs. This suppression was lost upon Treg-specific ablation of Stat3, a transcription factor critical for TH17 differentiation, and resulted in the development of a fatal intestinal inflammation. These findings suggest that Tregs adapt to their environment by engaging distinct effector response–specific suppression modalities upon activation of STAT proteins that direct the corresponding class of the immune response.

Interleukin-10 signaling in regulatory T cells is required for suppression of Th17 cell-mediated inflammation.

Effector CD4+ T cell subsets, whose differentiation is facilitated by distinct cytokine cues, amplify the corresponding type of inflammatory response. Regulatory T (Treg) cells integrate environmental cues to suppress particular types of inflammation. In this regard, STAT3, a transcription factor essential for T helper 17 (Th17) cell differentiation, is necessary for Treg cell-mediated control of Th17 cell responses. Here, we showed that anti-inflammatory interleukin-10 (IL-10), and not proinflammatory IL-6 and IL-23 cytokine signaling, endowed Treg cells with the ability to suppress pathogenic Th17 cell responses. Ablation of the IL-10 receptor in Treg cells resulted in selective dysregulation of Th17 cell responses and colitis similar to that observed in mice harboring STAT3-deficient Treg cells. Thus, Treg cells limit Th17 cell inflammation by serving as principal amplifiers of negative regulatory circuits operating in immune effector cells.

Extrathymic Generation of Regulatory T Cells in Placental Mammals Mitigates Maternal-Fetal Conflict

Regulatory T (Treg) cells, whose differentiation and function are controlled by X chromosome-encoded transcription factor Foxp3, are generated in the thymus (tTreg) and extrathymically (peripheral, pTreg), and their deficiency results in fatal autoimmunity. Here, we demonstrate that a Foxp3 enhancer, conserved noncoding sequence 1 (CNS1), essential for pTreg but dispensable for tTreg cell generation, is present only in placental mammals. CNS1 is largely composed of mammalian-wide interspersed repeats (MIR) that have undergone retrotransposition during early mammalian radiation. During pregnancy, pTreg cells specific to a model paternal alloantigen were generated in a CNS1-dependent manner and accumulated in the placenta. Furthermore, when mated with allogeneic, but not syngeneic, males, CNS1-deficient females showed increased fetal resorption accompanied by increased immune cell infiltration and defective remodeling of spiral arteries. Our results suggest that, during evolution, a CNS1-dependent mechanism of extrathymic differentiation of Treg cells emerged in placental animals to enforce maternal-fetal tolerance.

Transcription factor Foxp3 and its protein partners form a complex regulatory network

The transcription factor Foxp3 is indispensible for the differentiation and function of regulatory T cells (Treg cells). To gain insights into the molecular mechanisms of Foxp3-mediated gene expression, we purified Foxp3 complexes and explored their composition. Biochemical and mass-spectrometric analyses revealed that Foxp3 forms multiprotein complexes of 400–800 kDa or larger and identified 361 associated proteins, ∼30% of which were transcription related. Foxp3 directly regulated expression of a large proportion of the genes encoding its cofactors. Some transcription factor partners of Foxp3 facilitated its expression. Functional analysis of the cooperation of Foxp3 with one such partner, GATA-3, provided additional evidence for a network of transcriptional regulation afforded by Foxp3 and its associates to control distinct aspects of Treg cell biology.

Mouse regulatory DNA landscapes reveal global principles of cis-regulatory evolution

To study the evolutionary dynamics of regulatory DNA, we mapped >1.3 million deoxyribonuclease I–hypersensitive sites (DHSs) in 45 mouse cell and tissue types, and systematically compared these with human DHS maps from orthologous compartments. We found that the mouse and human genomes have undergone extensive cis-regulatory rewiring that combines branch-specific evolutionary innovation and loss with widespread repurposing of conserved DHSs to alternative cell fates, and that this process is mediated by turnover of transcription factor (TF) recognition elements. Despite pervasive evolutionary remodeling of the location and content of individual cis-regulatory regions, within orthologous mouse and human cell types the global fraction of regulatory DNA bases encoding recognition sites for each TF has been strictly conserved. Our findings provide new insights into the evolutionary forces shaping mammalian regulatory DNA landscapes. Mouse-to-human genomic comparisons illuminate conserved transcriptional programs despite regulatory rewiring. Rewiring the gene regulatory landscape DNAse I hypersensitive sites (DHSs) correlate with genomic locations that control where messenger RNA is to be produced. DHSs differ, depending on the cell type, developmental stage, and species. Viestra et al. compared mouse and human genome-wide DHS maps. Approximately one-third of the DHSs are conserved between the species, which separated approximately 550 million years ago. Most DHSs fell into tissue-specific cohorts; however, these were generally not conserved between the human and mouse. It seems that the majority of DHSs evolve because of changes in the sequence that gradually change how the region is regulated. Science, this issue p. 1007

Inflammation-induced repression of chromatin bound by the transcription factor Foxp3 in regulatory T cells

The transcription factor Foxp3 is indispensable for the ability of regulatory T (Treg) cells to suppress fatal inflammation. Here, we characterized the role of Foxp3 in chromatin remodeling and regulation of gene expression in actively suppressing Treg cells in an inflammatory setting. Although genome-wide Foxp3 occupancy of DNA regulatory elements was similar in resting and in vivo activated Treg cells, Foxp3-bound enhancers were poised for repression only in activated Treg cells. Following activation, Foxp3-bound sites showed reduced chromatin accessibility and selective H3K27 tri-methylation, which was associated with Ezh2 recruitment and downregulation of nearby gene expression. Thus, Foxp3 poises its targets for repression by facilitating formation of repressive chromatin in regulatory T cells upon their activation in response to inflammatory cues.The transcription factor Foxp3 is indispensable for the ability of regulatory T cells (Treg cells) to suppress fatal inflammation. Here we characterized the role of Foxp3 in chromatin remodeling and the regulation of gene expression in actively suppressive Treg cells in an inflammatory setting. Although genome-wide occupancy of regulatory elements in DNA by Foxp3 was similar in resting Treg cells and those activated in vivo, Foxp3-bound enhancer elements in the DNA were poised for repression only in activated Treg cells. Following activation, Foxp3-bound sites showed diminished accessibility of chromatin and selective deposition of histone H3 trimethylated at Lys27 (H3K27me3), which was associated with recruitment of the histone methyltransferase Ezh2 and downregulation of the expression of nearby genes. Thus, Foxp3 poises its targets for repression by facilitating the formation of repressive chromatin in Treg cells upon their activation in response to inflammatory cues.

Luteinizing Hormone-Releasing Hormone Enhances T Cell Recovery following Allogeneic Bone Marrow Transplantation

Posttransplant immunodeficiency, specifically a lack of T cell reconstitution, is a major complication of allogeneic bone marrow transplantation. This immunosuppression results in an increase in morbidity and mortality from infections and very likely contributes to relapse. In this study, we demonstrate that sex steroid ablation using leuprolide acetate, a luteinizing hormone-releasing hormone agonist (LHRHa), increases the number of lymphoid and myeloid progenitor cells in the bone marrow and developing thymocytes in the thymus. Although few differences are observed in the peripheral myeloid compartments, the enhanced thymic reconstitution following LHRHa treatment and allogeneic bone marrow transplantation leads to enhanced peripheral T cell recovery, predominantly in the naive T cell compartment. This results in an increase in T cell function in vivo and in vitro. Graft-versus-host-disease is not exacerbated by LHRHa treatment and graft-versus-tumor activity is maintained. Because LHRHa allows for reversible (and temporary) sex steroid ablation, has a strong safety profile, and has been clinically approved for diseases such as prostate and breast cancer, this drug treatment represents a novel therapeutic approach to reversal of thymic atrophy and enhancement of immunity following immunosuppression.

Patient HLA class I genotype influences cancer response to checkpoint blockade immunotherapy

HLA genotype affects response Immunotherapy works by activating the patients own immune system to fight cancer. For effective tumor killing, CD8+ T cells recognize tumor peptides presented by human leukocyte antigen class I (HLA-I) molecules. In humans, there are three major HLA-I genes (HLA-A, HLA-B, and HLA-C). Chowell et al. asked whether germline HLA-I genotype influences how T cells recognize tumor peptides and respond to checkpoint inhibitor immunotherapies (see the Perspective by Kvistborg and Yewdell). They examined more than 1500 patients and found that heterozygosity at HLA-I loci was associated with better survival than homozygosity for one or more HLA-I genes. Thus, specific HLA-I mutations could have implications for immune recognition and for the design of epitopes for cancer vaccines and immunotherapies. Science, this issue p. 582; see also p. 516 Human leukocyte antigen superfamilies predict immunotherapy response. CD8+ T cell–dependent killing of cancer cells requires efficient presentation of tumor antigens by human leukocyte antigen class I (HLA-I) molecules. However, the extent to which patient-specific HLA-I genotype influences response to anti–programmed cell death protein 1 or anti–cytotoxic T lymphocyte–associated protein 4 is currently unknown. We determined the HLA-I genotype of 1535 advanced cancer patients treated with immune checkpoint blockade (ICB). Maximal heterozygosity at HLA-I loci (“A,” “B,” and “C”) improved overall survival after ICB compared with patients who were homozygous for at least one HLA locus. In two independent melanoma cohorts, patients with the HLA-B44 supertype had extended survival, whereas the HLA-B62 supertype (including HLA-B*15:01) or somatic loss of heterozygosity at HLA-I was associated with poor outcome. Molecular dynamics simulations of HLA-B*15:01 revealed different elements that may impair CD8+ T cell recognition of neoantigens. Our results have important implications for predicting response to ICB and for the design of neoantigen-based therapeutic vaccines.

Localized sinonasal mucosal melanoma: Outcomes and associations with stage, radiotherapy, and positron emission tomography response.

Sinonasal mucosal melanoma is a rare neoplasm with a poor prognosis.

Locally Advanced and Unresectable Cutaneous Squamous Cell Carcinoma: Outcomes of Concurrent Cetuximab and Radiotherapy

Background. Advanced age and immune dysfunction are risk factors for cutaneous squamous cell carcinoma (cSCC) and often render patients with locally-advanced disease medically inoperable or surgically unresectable, but potentially curable with radiotherapy. Concurrent chemotherapy and radiotherapy may not be well tolerated in this population, but another systemic therapy may improve disease control. Objective. Determine the tolerance and efficacy of concurrent cetuximab and radiotherapy (CRT) for patients with locally advanced and unresectable cSCC. Methods. Retrospective analysis of 12 patients treated with CRT for locally advanced and unresectable cSCC. Results. Patients were elderly and 75% had moderate-to-severe comorbidities, while 42% had immune dysfunction. Grades 3-4 adverse events were noted in 83% of patients; 67% required hospital admission for adverse events. Complete and partial response was noted in 36% and 27% (response rate, 64%). Stable and progressive disease was noted in 3 and 1 patients, respectively (disease control rate, 91%). Median progression-free and overall survival were 6.4 and 8.0 months, respectively. Limitations. Retrospective small-cohort, single-institution analysis. Conclusion. Patients selected for CRT were elderly, with comorbidities and immune dysfunction, but treatment responses were observed. Patients selected for this treatment approach have a poor prognosis with limited capacity for therapy; more effective treatment is needed.