SUMMARY Diffuse large B cell lymphoma (DLBCL) is the most common form of blood cancer and is characterized by a striking degree of genetic and clinical heterogeneity. This heterogeneity poses a major barrier to understanding the genetic basis of the disease and its response to therapy. Here, we performed an integrative analysis of whole exome sequencing and transcriptome sequencing in a cohort of 1001 DLBCL patients to comprehensively define the landscape of 150 genetic drivers of the disease. We characterized the functional impact of these genes using an unbiased CRISPR screen of DLBCL cell lines to define oncogenes that promote cell growth. A prognostic model comprising these genetic alterations outperformed current established methods: cell of origin, the International Prognostic Index comprising clinical variables, and dual MYC and BCL2 expression. These results comprehensively define the genetic drivers and their functional roles in DLBCL to identify new therapeutic opportunities in the disease.

CD4+ T helper cells acquire effector phenotypes that promote specialized inflammatory responses. We show that the ETS family transcription factor, PU.1 was required for the development of an interleukin 9 (IL-9)-secreting subset of TH cells. Decreasing PU.1 expression either by conditional deletion in murine T cells or siRNA in human T cells impaired IL-9 production, while ectopic PU.1 expression promoted IL-9 production. Mice with PU.1-deficient T cells developed normal TH2 responses in vivo, but exhibited attenuated allergic pulmonary inflammation corresponding to decreased Il9 and chemokine expression in peripheral T cells and in lungs as compared to wild-type mice. Together, these data suggest a critical role for PU.1 in generating the TH9 phenotype and in the development of allergic inflammation.

T helper 9 (Th9) cells are specialized for the production of IL-9, promote allergic inflammation in mice, and are associated with allergic disease in humans. It has not been determined whether Th9 cells express a characteristic transcriptional signature. In this study, we performed microarray analysis to identify genes enriched in Th9 cells compared with other Th subsets. This analysis defined a transcriptional regulatory network required for the expression of a subset of Th9-enriched genes. The activator protein 1 (AP1) family transcription factor BATF (B cell, activating transcription factor-like) was among the genes enriched in Th9 cells and was required for the expression of IL-9 and other Th9-associated genes in both human and mouse T cells. The expression of BATF was increased in Th9 cultures derived from atopic infants compared with Th9 cultures from control infants. T cells deficient in BATF expression had a diminished capacity to promote allergic inflammation compared with wild-type controls. Moreover, mouse Th9 cells ectopically expressing BATF were more efficient at promoting allergic inflammation than control transduced cells. These data indicate that BATF is a central regulator of the Th9 phenotype and contributes to the development of allergic inflammation.

Summary Transcriptional regulatory networks direct the development of specialized cell types. The transcription factors Stat4 and T-bet are required for the development of T helper 1 cells, although the hierarchy of activity by these factors has not been clearly defined. In this report we show that these factors are not in a linear pathway and that each factor plays a unique role in programming chromatin architecture for Th1 gene expression, with subsets of genes depending on Stat4, T-bet, or both for expression in Th1 cells. T-bet is not able to transactivate expression of Stat4-dependent genes in the absence of endogenous Stat4 expression. Thus, T-bet requires Stat4 to achieve complete Th1 fate determination.

IL-4 promotes the development of Th2 cells and allergic inflammation. In atopic dermatitis lesions, IL-4 decreases the expression of multiple genes associated with innate defense, including genes in the epidermal differentiation complex (EDC) that regulate epidermal barrier function. However, it is not clear whether IL-4 also contributes to homeostatic control of EDC genes. In this report, we demonstrate that expression of EDC genes and barrier function is increased in the absence of endogenous IL-4. Mice that express a constitutively active Stat6 (Stat6VT) are prone to the development of allergic skin inflammation and have decreased expression of EDC genes. IL-4 deficiency protects Stat6VT transgenic mice from the development of allergic skin inflammation and decreased recovery time in barrier function following skin irritation, with a concomitant increase in EDC gene expression. These data suggest that IL-4 plays an important role in regulating epidermal homeostasis and innate barrier function.

Signal Transducer and Activator of Transcription (STAT) family members direct the differentiation of T helper cells, with specific STAT proteins promoting distinct effector subsets. STAT6 is required for the development of T helper 2 (Th2) cells, whereas STAT3 promotes differentiation of Th17 and follicular helper T cell subsets. We demonstrated that STAT3 was also activated during Th2 cell development and was required for the expression of Th2-cell associated cytokines and transcription factors. STAT3 bound directly to Th2-cell associated gene loci and was required for the ability of STAT6 to bind target genes. In vivo, STAT3-deficiency in T cells eliminated the allergic inflammation in mice sensitized and challenged with ovalbumin, or transgenic for constitutively active STAT6. Thus, STAT3 cooperates with STAT6 in promoting Th2 cell development. These results demonstrate that differentiating T helper cells integrate multiple STAT protein signals during Th2 cell development.

Thymic stromal lymphopoietin (TSLP) is an epithelial cell derived cytokine important for the initiation and development of T helper (Th2) cell-mediated allergic inflammation. In this study, we identified a positive association between interleukin-9 (IL-9) and TSLP concentration in the serum of infants with atopic dermatitis. In primary cell cultures, the addition of TSLP led to an increase in IL-9 production from human and mouse Th9 cells, and induced an increase in Signal Transducer and Activator of Transcription 5 (STAT5) activation and binding to the Il9 promoter. In vivo, use of an adoptive transfer model demonstrated that TSLP promoted IL-9-dependent, Th9 cell-induced allergic inflammation by acting directly on T cells. Moreover, transgenic expression of TSLP in the lung stimulated IL-9 production in vivo, and anti-IL-9 treatment attenuated TSLP-induced airway inflammation. Together, our results demonstrate that TSLP promotes Th9 cell differentiation and function, and define a requirement for IL-9 in TSLP-induced allergic inflammation.

Background IL-9 is important for the growth and survival of mast cells. IL-9 is produced by T cells, NKT cells, mast cells, eosinophils, and innate lymphoid cells, although the cells required for mast cell accumulation during allergic inflammation remain undefined. Objective To elucidate the role of Th9 cells in promoting mast cell accumulation in models of allergic lung inflammation. Methods Adoptive transfer of OVA-specific Th2 and Th9 cells was used to assess the ability of each subset to mediate mast cell accumulation in tissues. Mast cell accumulation was assessed in wild type mice and mice with PU.1-deficient T cells subjected to acute and chronic models of allergic inflammation. Results Adoptive transfer experiments demonstrated that recipients of Th9 cells had significantly higher mast cell accumulation and expression of mast cell proteases as compared to control or Th2 recipients. Mast cell accumulation was dependent on IL-9, but not IL-13, cytokine required for many aspects of allergic inflammation. In models of acute and chronic allergic inflammation, decreased IL-9 levels in mice with PU.1-deficient T cells corresponded to diminished tissue mast cell numbers and expression of mast cell proteases. Mice with PU.1-deficient T cells have defects in IL-9 production from CD4+ T cells, but not NKT cells or innate lymphoid cells, suggesting a T helper cell-dependent phenotype. Rag1−/− mice subjected to a chronic model of allergic inflammation displayed reduced mast cell infiltration comparable to accumulation in mice with PU.1-deficient T cells, emphasizing the importance of IL-9 produced by T cells in mast cell recruitment. Conclusion Th9 cells are a major source of IL-9 in models of allergic inflammation and play an important role in mast cell accumulation and activation.