Current T cell differentiation models invoke separate T helper 2 (Th2) and Th1 cell lineages governed by the lineage-specifying transcription factors GATA-3 and T-bet. However, knowledge on the plasticity of Th2 cell lineage commitment is limited. Here we show that infection with Th1 cell-promoting lymphocytic choriomeningitis virus (LCMV) reprogrammed otherwise stably committed GATA-3(+) Th2 cells to adopt a GATA-3(+)T-bet(+) and interleukin-4(+)interferon-gamma(+) "Th2+1" phenotype that was maintained in vivo for months. Th2 cell reprogramming required T cell receptor stimulation, concerted type I and type II interferon and interleukin-12 signals, and T-bet. LCMV-triggered T-bet induction in adoptively transferred virus-specific Th2 cells was crucial to prevent viral persistence and fatal immunopathology. Thus, functional reprogramming of unfavorably differentiated Th2 cells may facilitate the establishment of protective immune responses. Stable coexpression of GATA-3 and T-bet provides a molecular concept for the long-term coexistence of Th2 and Th1 cell lineage characteristics in single memory T cells.

The stable lineage commitment of naïve T helper cells to a hybrid Th1/2 phenotype reveals the cell-intrinsic reconciliation of two opposing T cell differentiation programs and provides a self-limiting mechanism to dampen immunopathology.

During infection, the release of damage-associated molecular patterns, so-called "alarmins," orchestrates the immune response. The alarmin IL-33 plays a role in a wide range of pathologies. Upon release, IL-33 signals through its receptor ST2, which reportedly is expressed only on CD4 + T cells of the Th2 and regulatory subsets.Here we show that Th1 effector cells also express ST2 upon differentiation in vitro and in vivo during lymphocytic choriomeningitis virus (LCMV) infection. The expression of ST2 on Th1 cells was transient, in contrast to constitutive ST2 expression on Th2 cells, and marked highly activated effector cells. ST2 expression on virusspecific Th1 cells depended on the Th1-associated transcription factors T-bet and STAT4. ST2 deficiency resulted in a T-cell-intrinsic impairment of LCMV-specific Th1 effector responses in both mixed bone marrow-chimeric mice and adoptive cell transfer experiments. ST2-deficient virus-specific CD4 + T cells showed impaired expansion, Th1 effector differentiation, and antiviral cytokine production. Consequently, these cells mediated little virus-induced immunopathology. Thus, IL-33 acts as a critical and direct cofactor to drive antiviral Th1 effector cell activation, with implications for vaccination strategies and immunotherapeutic approaches.IL-33 | ST2 | CD4 T cell | Th1 cell | virus infection I ntercellular signaling molecules, such as cytokines and damageassociated molecular patterns (DAMPs), are essential for the induction and amplification of immune responses. DAMPs are multifunctional host proteins that indicate tissue damage. They are also referred to as alarmins, because they serve as early warning signals to activate innate and adaptive immune responses (1). The alarmin IL-33, a member of the IL-1 family, is constitutively expressed in the nucleus of endothelial and epithelial cells and is released during tissue damage and necrosis (2). Upon release, IL-33 mediates its activity through a heterodimeric cell surface receptor consisting of the ubiquitous IL-1R accessory protein (IL1RAcP) and the more selectively expressed receptor ST2, also known as T1 and IL-1RL1 (3, 4). The intracellular signaling pathway of this receptor complex includes the recruitment of MyD88 and leads to the activation of NF-κB, ERK, p38, and JNK pathways (2).IL-33 acts on a wide range of immune cells, but has been implicated predominantly in Th2-associated immune responses (5). Nonetheless, this categorization has recently been challenged by reports describing IL-33 as an enhancer of IFN-γ production by iNKT cells, NK cells, and CD8+ T cells (6-8). In addition, IL-33 signaling is important for the activation and functionality of cytotoxic CD8 + T cells during viral infection (9). In the present work, we studied a potential impact of IL-33 on Th1 cell responses in vitro and in vivo. In the course of infection with LCMV, which potently induces Th1-differentiated effector CD4 + T cells (10), we found that many of the virus-specific CD4 + T cells expressed the IL-33 receptor ST2. ST2 ex...

Combining experiments on primary T cells and mathematical modeling, we characterized the stochastic expression of the interleukin-4 cytokine gene in its physiologic context, showing that a two-step model of transcriptional regulation acting on chromatin rearrangement and RNA polymerase recruitment accounts for the level, kinetics, and population variability of expression.A rate-limiting step upstream of transcription initiation, but occurring at the level of an individual allele, controls whether the interleukin-4 gene is expressed during antigenic stimulation, suggesting that the observed stochasticity of expression is linked to the dynamics of chromatin rearrangement.The computational analysis predicts that the probability to re-express an interleukin-4 gene that has been expressed once is transiently increased. In support, we experimentally demonstrate a short-term memory for interleukin-4 expression at the predicted time scale of several days.The model provides a unifying framework that accounts for both graded and binary modes of gene regulation. Graded changes in expression level can be achieved by controlling transcription initiation, whereas binary regulation acts at the level of chromatin rearrangement and is targeted during the differentiation of T cells that specialize in interleukin-4 production.

SUMMARY The probabilistic expression of cytokine genes in differentiated T helper (Th) cell populations remains ill defined. By single-cell analyses and mathematical modeling, we show that one stimulation featured stable cytokine nonproducers as well as stable producers with wide cell-to-cell variability in the magnitude of expression. Focusing on interferon-γ (IFN-γ) expression by Th1 cells, mathematical modeling predicted that this behavior reflected different cell-intrinsic capacities and not mere gene-expression noise. In vivo, Th1 cells sort purified by secreted IFN-γ amounts preserved a quantitative memory for both probability and magnitude of IFN-γ re-expression for at least 1 month. Mechanistically, this memory resulted from quantitatively distinct transcription of individual alleles and was controlled by stable expression differences of the Th1 cell lineage-specifying transcription factor T-bet. Functionally, Th1 cells with graded IFN-γ production competence differentially activated Salmonella-infected macrophages for bacterial killing. Thus, individual Th cells commit to produce distinct amounts of a given cytokine, thereby generating functional intrapopulation heterogeneity.