Cytotoxic Programming of CD4+ T Cells Is Regulated by Opposing Actions of the Related Transcription Factors Eos and Aiolos.

In contrast to the "helper" activities of most CD4+ T effector subsets, CD4+ cytotoxic T lymphocytes (CD4-CTLs) perform functions normally associated with CD8+ T and NK cells. Specifically, CD4-CTLs secrete cytotoxic molecules and directly target and kill compromised cells in an MHC class II-restricted fashion. The functions of these cells have been described in diverse immunological contexts, including their ability to provide protection during antiviral and antitumor responses, as well as being implicated in autoimmunity. Despite their significance to human health, the complete mechanisms that govern their programming remain unclear. In this article, we identify the Ikaros zinc finger transcription factor Eos (Ikzf4) as a positive regulator of CD4-CTL differentiation during murine immune responses against influenza virus infection. We find that the frequency of Eos+ cells is elevated in lung CD4-CTL populations and that the cytotoxic gene program is compromised in Eos-deficient CD4+ T cells. Consequently, we observe a reduced frequency and number of lung-residing, influenza virus-responsive CD4-CTLs in the absence of Eos. Mechanistically, we determine that this is due, at least in part, to reduced expression of IL-2 and IL-15 cytokine receptor subunits on the surface of Eos-deficient CD4+ T cells, both of which support the CD4-CTL program. Finally, we find that Aiolos, a related Ikaros family member and known CD4-CTL antagonist, represses Eos expression by antagonizing STAT5-dependent activation of the Ikzf4 promoter. Collectively, our findings reveal a mechanism wherein Eos and Aiolos act in opposition to regulate cytotoxic programming of CD4+ T cells.

Eos Promotes TH2 Differentiation by Interacting with and Propagating the Activity of STAT5.

The Ikaros zinc-finger transcription factor Eos has largely been associated with sustaining the immunosuppressive functions of regulatory T cells. Paradoxically, Eos has more recently been implicated in promoting proinflammatory responses in the dysregulated setting of autoimmunity. However, the precise role of Eos in regulating the differentiation and function of effector CD4+ T cell subsets remains unclear. In this study, we find that Eos is a positive regulator of the differentiation of murine CD4+ TH2 cells, an effector population that has been implicated in both immunity against helminthic parasites and the induction of allergic asthma. Using murine in vitro TH2 polarization and an in vivo house dust mite asthma model, we find that EosKO T cells exhibit reduced expression of key TH2 transcription factors, effector cytokines, and cytokine receptors. Mechanistically, we find that the IL-2/STAT5 axis and its downstream TH2 gene targets are one of the most significantly downregulated pathways in Eos-deficient cells. Consistent with these observations, we find that Eos forms, to our knowledge, a novel complex with and supports the tyrosine phosphorylation of STAT5. Collectively, these data define a regulatory mechanism whereby Eos propagates STAT5 activity to facilitate TH2 cell differentiation.

Integrated STAT3 and Ikaros Zinc Finger Transcription Factor Activities Regulate Bcl-6 Expression in CD4+ Th Cells.

B cell lymphoma-6 (Bcl-6) is a transcriptional repressor that is required for the differentiation of T follicular helper (TFH) cell populations. Currently, the molecular mechanisms underlying the transcriptional regulation of Bcl-6 expression are unclear. In this study, we have identified the Ikaros zinc finger transcription factors Aiolos and Ikaros as novel regulators of Bcl-6. We found that increased expression of Bcl-6 in CD4+ Th cell populations correlated with enhanced enrichment of Aiolos and Ikaros at the Bcl6 promoter. Furthermore, overexpression of Aiolos or Ikaros, but not the related family member Eos, was sufficient to induce Bcl6 promoter activity. Intriguingly, STAT3, a known Bcl-6 transcriptional regulator, physically interacted with Aiolos to form a transcription factor complex capable of inducing the expression of Bcl6 and the TFH-associated cytokine receptor Il6ra Importantly, in vivo studies revealed that the expression of Aiolos was elevated in Ag-specific TFH cells compared with that observed in non-TFH effector Th cells generated in response to influenza infection. Collectively, these data describe a novel regulatory mechanism through which STAT3 and the Ikaros zinc finger transcription factors Aiolos and Ikaros cooperate to regulate Bcl-6 expression.

Rapid assembly of SARS-CoV-2 genomes reveals attenuation of the Omicron BA.1 variant through NSP6.

Although the SARS-CoV-2 Omicron variant (BA.1) spread rapidly across the world and effectively evaded immune responses, its viral fitness in cell and animal models was reduced. The precise nature of this attenuation remains unknown as generating replication-competent viral genomes is challenging because of the length of the viral genome (30kb). Here, we designed a plasmid-based viral genome assembly and resc ue strategy (pGLUE) that constructs complete infectious viruses or noninfectious subgenomic replicons in a single ligation reaction with >80% efficiency. Fully sequenced replicons and infectious viral stocks can be generated in 1 and 3 weeks, respectively. By testing a series of naturally occurring viruses as well as Delta-Omicron chimeric replicons, we show that Omicron nonstructural protein 6 harbors critical attenuating mutations, which dampen viral RNA replication and reduce lipid droplet consumption. Thus, pGLUE overcomes remaining barriers to broadly study SARS-CoV-2 replication and reveals deficits in nonstructural protein function underlying Omicron attenuation.

Rapid assembly of SARS-CoV-2 genomes reveals attenuation of the Omicron BA.1 variant through NSP6.

Although the SARS-CoV-2 Omicron variant (BA.1) spread rapidly across the world and effectively evaded immune responses, its viral fitness in cell and animal models was reduced. The precise nature of this attenuation remains unknown as generating replication-competent viral genomes is challenging because of the length of the viral genome (~30 kb). Here, we present a plasmid-based viral genome assembly and rescue strategy (pGLUE) that constructs complete infectious viruses or noninfectious subgenomic replicons in a single ligation reaction with >80% efficiency. Fully sequenced replicons and infectious viral stocks can be generated in 1 and 3 weeks, respectively. By testing a series of naturally occurring viruses as well as Delta-Omicron chimeric replicons, we show that Omicron nonstructural protein 6 harbors critical attenuating mutations, which dampen viral RNA replication and reduce lipid droplet consumption. Thus, pGLUE overcomes remaining barriers to broadly study SARS-CoV-2 replication and reveals deficits in nonstructural protein function underlying Omicron attenuation.

Taking cues from convalescence to improve vaccines against hepatitis C virus.

Hepatitis C virus (HCV) infection remains a worldwide public health issue despite direct-acting antivirals. A substantial proportion of infected individuals (15%-45%) spontaneously clear repeated HCV infections with genetically different viruses by generating broadly neutralizing antibodies (bNAbs). However, translating this response into an effective vaccine strategy has been unsuccessful. In this issue of the JCI, Frumento and colleagues report on their study of bNAb evolution longitudinally in convalescent individuals with repeated infections. Using pseudotyped viruses, well-characterized monoclonal antibodies, and complex modeling, the authors show that multiple exposures to antigenically related, antibody-sensitive viral envelope proteins induced potent bNAbs. This work provides valuable insight into the best strategies for developing HCV vaccines in the future that may successfully reproduce the immunity induced during natural exposures.

In Vitro Differentiation of Effector CD4+ T Helper Cell Subsets.

CD4+ T "helper" cells are key orchestrators of adaptive immune responses. Upon activation, naïve CD4+ T cells are capable of differentiating into a number of effector subsets that perform distinct immune functions. These subsets include T helper 1 (TH1), TH2, TH9, TH17, TH22, T follicular helper (TFH), and regulatory T cell (TREG) populations. The differentiation of these subsets is dependent, in large part, on the coordinated interplay between signals from the extracellular cytokine environment and downstream transcriptional networks. The use of in vitro T helper cell culture systems has been extensively employed to aid in the elucidation of the molecular mechanisms that govern the differentiation of each effector subset. Here, we provide a detailed summary of the differentiation conditions that are utilized to generate effector CD4+ T cell populations in vitro.

Ikaros Zinc Finger Transcription Factors: Regulators of Cytokine Signaling Pathways and CD4+ T Helper Cell Differentiation.

CD4+ T helper cells are capable of differentiating into a number of effector subsets that perform diverse functions during adaptive immune responses. The differentiation of each of these subsets is governed, in large part, by environmental cytokine signals and the subsequent activation of downstream, cell-intrinsic transcription factor networks. Ikaros zinc finger (IkZF) transcription factors are known regulators of immune cell development, including that of CD4+ T cell subsets. Over the past decade, members of the IkZF family have also been implicated in the differentiation and function of individual T helper cell subsets, including T helper 1 (TH1), TH2, TH17, T follicular (TFH), and T regulatory (TREG) cells. Now, an increasing body of literature suggests that the distinct cell-specific cytokine environments responsible for the development of each subset result in differential expression of IkZF factors across T helper populations. Intriguingly, recent studies suggest that IkZF members influence T helper subset differentiation in a feed-forward fashion through the regulation of these same cytokine-signaling pathways. Here, we review the increasingly prominent role for IkZF transcription factors in the differentiation of effector CD4+ T helper cell subsets.

IL-12 signaling drives the differentiation and function of a TH1-derived TFH1-like cell population.

CD4+ T follicular helper (TFH) cells provide help to B cells and promote antibody-mediated immune responses. Increasing evidence supports the existence of TFH populations that secrete cytokines typically associated with the effector functions of other CD4+ T cell subsets. These include T helper 1 (TH1)-biased TFH (TFH1) cells that have recognized roles in both immune responses to pathogens and also the pathogenesis of autoimmune disease. Given their apparent importance to human health, there is interest in understanding the mechanisms that regulate TFH1 cell formation and function. However, their origin and the molecular requirements for their differentiation are unclear. Here, we describe a population of murine TH1-derived, TFH1-like cells that express the chemokine receptor Cxcr3 and produce both the TH1 cytokine interferon-γ and the TFH-associated cytokine interleukin-21 (IL-21). Furthermore, these TFH1-like cells promote B cell activation and antibody production at levels indistinguishable from conventional IL-6-derived TFH-like cells. Regarding their regulatory requirements, we find that IL-12 signaling is necessary for the differentiation and function of this TFH1-like cell population. Specifically, IL-12-dependent activation of STAT4, and unexpectedly STAT3, promotes increased expression of IL-21 and the TFH lineage-defining transcription factor Bcl-6 in TFH1-like cells. Taken together, these findings provide insight into the potential origin and differentiation requirements of TFH1 cells.

PEDF inhibits pancreatic tumorigenesis by attenuating the fibro-inflammatory reaction.

Pancreatic cancer is characterized by a pronounced fibro-inflammatory reaction that has been shown to contribute to cancer progression. Previous reports have demonstrated that pigment epithelium-derived factor (PEDF) has potent tumor suppressive effects in pancreatic cancer, though little is known about the mechanisms by which PEDF limits pancreatic tumorigenesis. We therefore employed human specimens, as well as mouse and in vitro models, to explore the effects of PEDF upon the pancreatic microenvironment. We found that PEDF expression is decreased in human pancreatic cancer samples compared to non-malignant tissue. Furthermore, PEDF-deficient patients displayed increased intratumoral inflammation/fibrosis. In mice, genetic ablation of PEDF increased cerulein-induced inflammation and fibrosis, and similarly enhanced these events in the background of oncogenic KRAS. In vitro, recombinant PEDF neutralized macrophage migration as well as inhibited macrophage-induced proliferation of tumor cells. Additionally, recombinant PEDF suppressed the synthesis of pro-inflammatory/pro-fibrotic cytokines both in vivo and in vitro, and reduced collagen I deposition and TGFß synthesis by pancreatic stellate cells, consistent with reduced fibrosis. Combined, our results demonstrate that PEDF limits pancreatic cancer progression by attenuating the fibro-inflammatory reaction, and makes restoration of PEDF signaling a potential therapeutic approach to study in pancreatic cancer.

Polarization of the vacuolar adenosine triphosphatase delineates a transition to high-grade pancreatic intraepithelial neoplasm lesions.

OBJECTIVES: A functional vacuolar adenosine triphosphatase (v-ATPase) complex regulates canonical Wnt/ß-catenin signaling. The goal of this study was to identify the distribution of the v-ATPase in human and murine models of pancreatic intraepithelial neoplasms (PanINs) and assess its role in Wnt/ß-catenin signaling. METHODS: We evaluated the immunolabeling pattern of the v-ATPase in human PanIN specimens and murine PanIN-1 and PanIN-2 lesions obtained from Ptf1a(Cre/+); LSL-Kras(G12D) mice. Wnt/ß-catenin signaling was interrogated in primary PanIN cells by examining the phosphorylated levels of its surface coreceptor, low-density lipoprotein receptor-related protein-6 (LRP6), and its intracellular effector, nonphosphorylated ß-catenin. The response of primary PanIN cells to epidermal growth factor (EGF) was assessed in the absence and presence of the v-ATPase inhibitor, concanamycin. RESULTS: In advanced (PanIN-2), but not early (PanIN-1), lesions, the v-ATPase assumed a polarized phenotype. Blocking the v-ATPase disrupted Wnt/ß-catenin signaling in primary PanIN cells despite significantly higher levels of the total and activated Wnt cell surface coreceptor, LRP6. Vacuolar adenosine triphosphatase blockade significantly decreased the total and activated levels of EGF receptor, a determinant of PanIN progression. The activation of EGF receptor and its intracellular mediator, p44/42 mitogen-activated protein kinase, was also reduced by v-ATPase blockade. This led to diminished proliferation in response to EGF ligand. CONCLUSIONS: The v-ATPase regulates Wnt/ß-catenin and EGF receptor signaling in PanINs.