Cell transfer experiments complement the rigorous investigation of antiviral and antitumor functions of natural killer (NK) cells. Success in these endeavors is enhanced by expansion of small numbers of input NK cells driven by viral antigens or homeostatic proliferation in immunodeficient hosts. In contrast, analysis of other NK-cell functions, including immunoregulation, are non-proliferative and require an intact immune system in recipient mice. We reveal poor persistence of conventional congenic (CD45.1) BoyJ NK cells following adoptive transfer in comparison to CRISPR-generated CD45.1+ (JAXBoy) NK cells. Reciprocal transfers between C57BL/6 and JAXBoy mice substantially improve seeding and maintenance of donor NK cells. Using this system, we confirm that CXCR3 re-positions NK cells in the white pulp of the spleen after infection, which is vital for immunoregulation. Moreover, we discovered that the transcription factor ASCL2 is required for recruitment of NK cells into the spleen and white pulp. These results provide improved tools and novel insights into NK cell biology.

IntroductionAdjuvants added to subunit vaccines augment antigen-specific immune responses. One mechanism of adjuvant action is activation of pattern recognition receptors (PRRs) on innate immune cells. Bordetella colonization factor A (BcfA); an outer membrane protein with adjuvant function, activates TH1/TH17-polarized immune responses to protein antigens from Bordetella pertussis and SARS CoV-2. Unlike other adjuvants, BcfA does not elicit a TH2 response.MethodsTo understand the mechanism of BcfA-driven TH1/TH17 vs. TH2 activation, we screened PRRs to identify pathways activated by BcfA. We then tested the role of this receptor in the BcfA-mediated activation of bone marrow-derived dendritic cells (BMDCs) using mice with germline deletion of TLR4 to quantify upregulation of costimulatory molecule expression and cytokine production in vitro and in vivo. Activity was also tested on human PBMCs.ResultsPRR screening showed that BcfA activates antigen presenting cells through murine TLR4. BcfA-treated WT BMDCs upregulated expression of the costimulatory molecules CD40, CD80, and CD86 and produced IL-6, IL-12/23 p40, and TNF-α while TLR4 KO BMDCs were not activated. Furthermore, human PBMCs stimulated with BcfA produced IL-6. BcfA-stimulated murine BMDCs also exhibited increased uptake of the antigen DQ-OVA, supporting a role for BcfA in improving antigen presentation to T cells. BcfA further activated APCs in murine lungs. Using an in vitro TH cell polarization system, we found that BcfA-stimulated BMDC supernatant supported TFH and TH1 while suppressing TH2 gene programming.ConclusionsOverall, these data provide mechanistic understanding of how this novel adjuvant activates immune responses.

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.

CD4+ T helper 1 (Th1) cells coordinate adaptive immune responses to intracellular pathogens, including viruses. Key to this function is the ability of Th1 cells to migrate within secondary lymphoid tissues, as well as to sites of inflammation, which relies on signals received through the chemokine receptor CXCR3. CXCR3 expression is driven by the Th1 lineage-defining transcription factor T-bet and the cytokine-responsive STAT family members STAT1 and STAT4. Here, we identify the Ikaros zinc finger (IkZF) transcription factor Aiolos (Ikzf3) as an additional positive regulator of CXCR3 both in vitro and in vivo using a murine model of influenza virus infection. Mechanistically, we found that Aiolos-deficient CD4+ T cells exhibited decreased expression of key components of the IFN-γ/STAT1 signaling pathway, including JAK2 and STAT1. Consequently, Aiolos deficiency resulted in decreased levels of STAT1 tyrosine phosphorylation and reduced STAT1 enrichment at the Cxcr3 promoter. We further found that Aiolos and STAT1 formed a positive feedback loop via reciprocal regulation of each other downstream of IFN-γ signaling. Collectively, our study demonstrates that Aiolos promotes CXCR3 expression on Th1 cells by propagating the IFN-γ/STAT1 cytokine signaling pathway.

CD4 +cytotoxic T lymphocytes, or CD4-CTLs, comprise a CD4 +subset capable of performing functions normally observed in CD8 +T and Natural Killer cells. CD4-CTLs play critical roles in many immune contexts, including protective anti-viral responses to viruses such as SARS-CoV-2 and influenza. Despite their well-documented importance, the complete mechanisms that underlie their formation remain unclear. Here, we define the transcription factor Aiolos as a novel repressor of CD4-CTL differentiation. We find that Aiolos deficiency results in increased CD4-CTL responses in lungs of influenza-infected mice, as assessed by elevated expression of Granzyme B and Perforin, as well as the CTL marker NKG2A/C/E. We further find that Aiolos-deficient CD4-CTLs exhibit increased expression of transcription factors associated with cytotoxic programming, including Eomes and Blimp-1. Mechanistically, we find that Aiolos-deficient cells have a heightened sensitivity to IL-2/STAT5 signaling due to enhanced expression of IL-2 receptor subunits. This translates into increased chromatin accessibility and STAT5 association at regulatory regions of hallmark CD4-CTL genes in the absence of Aiolos. Consistent with these data, in silico analyses demonstrate that STAT5 DNA-binding motifs are significantly enriched at these same regions. Intriguingly, the STAT5 motif partially overlaps with that of the core Aiolos DNA binding motif, suggesting that Aiolos may function to broadly antagonize STAT5 activity throughout the genome. Collectively, this work establishes Aiolos as a novel repressor of cytotoxic programming in CD4 +T cells and highlights its potential as a therapeutic target for enhancing anti-viral immunity. This work was supported by grants from The National Institutes of Health AI134972 and AI127800 to K.J.O, AI156411 to P.L.C. K.A.R. is supported by funding through The Ohio State University College of Medicine Advancing Research in Infection and Immunity Fellowship Program. J.A.T. and S.P. are supported through funding from the Infectious Disease Institute T32 in Host-Microbe Interactions fellowship.

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(+) T(H)2 cells, an effector population that has been implicated in both immunity against helminthic parasites and the induction of allergic asthma. Using murine in vitro T(H)2 polarization and an in vivo house dust mite asthma model, we find that Eos(KO) T cells exhibit reduced expression of key T(H)2 transcription factors, effector cytokines, and cytokine receptors. Mechanistically, we find that the IL-2/STAT5 axis and its downstream T(H)2 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 T(H)2 cell differentiation.

The Ikaros zinc finger transcription factor Eos has largely been associated with chromatin regulation promoting immunosuppressive regulatory T cells. However, Eos’ role in the differentiation and function of pro-inflammatory T cells has remained unclear. Surprisingly, our work reveals that Eos is a positive regulator of CD4 +T helper 2 (T H2) cells—effector T cells that participate in anti-helminthic immunity but are also implicated in inducing allergic asthma. Using in vitro-generated T H2 cells and an in vivo allergic asthma mouse model, we found that Eos-deficient T cells had reduced gene and protein expression of critical T H2 transcription factors (including the lineage-defining transcription factor Gata3), effector cytokines, and differentiation receptors. Among the various T H2-polarizing pathways, the IL-2/STAT5 axis and its downstream T H2 gene targets emerged as one of the most significantly downregulated networks in Eos deficiency. Using in vitro-generated T H2 cells and overexpression of Eos zinc-finger-domain mutants, we discovered that Eos forms a novel complex with and promotes the tyrosine-phosphorylated activation of STAT5. Additionally, we showed that components of the IL-2/STAT5 pathway participate in a feed-forward loop to promote Eos expression in T H2 cells, further supporting Eos’ regulatory connection with the IL-2/STAT5 axis. Together, these data define a novel mechanism whereby Eos mediates IL-2/STAT5 activity to facilitate T H2 differentiation. This work is significant, as its findings reframe our understanding of Eos’ role in T cell differentiation. K.J.O. is supported by a grant from The National Institutes of Health (NIH) AI134972, as well as from The Ohio State University College of Medicine and The Ohio State University Comprehensive Cancer Center. J.A.T. is supported by funding through the Susan Huntington Dean’s Distinguished University Fellowship and the NIH T32 “Interdisciplinary Program in Microbe-Host Biology” pre-doctoral fellowship administered through the OSU Infectious Diseases Institute and OSU Department of Microbial Infection and Immunity. K.M.G. is supported by NIH grant 1R01ES028829-01A1. L.M.C. and K.J.O. were supported in part by the Jeffress Trust Awards Program in Interdisciplinary Research. K.A.R. is supported by funding through The Ohio State University College of Medicine Advancing Research in Infection and Immunity Fellowship Program.

The X-chromosome linked RNA helicase DDX3X plays multiple roles in RNA metabolism and antiviral interferon responses. Some viruses, including SARS-CoV-2, usurp the function of DDX3X to support viral genome replication and evasion of antiviral interferon. These findings have led to development of DDX3X inhibitors as potential antiviral agents. However, somatic loss of function mutations in this helicase are linked to neurological disease and malignancies, including a form of non-Hodgkin’s lymphoma initiated in the natural killer (NK) cell lineage. Given the critical role of NK cells in antiviral and antitumor immunity, we sought to determine how inhibition of DDX3X could affect NK-cell biology. Constitutive (Ncr1-iCre) or inducible (Ncr1-iCreER T2) deletion of Ddx3x in Ncr1-expressing cells (i.e. NK cells) in mice resulted in a complete loss of peripheral NK cells. Addition of the DDX3X inhibitor RK-33 to in vitro cultures of NK cells resulted in a similar loss of cellularity, while deletion of Ddx3x blocked development of NK cells from progenitor cells in an in vitro culture on stromal cells. Preliminary analyses suggest that loss of DDX3X results in rapid cell death of committed NK cells. Partial preservation of early stages of NK-cell development in the bone marrow of male but not female Ncr1 DDdx3xsuggests that the Y-chromosome DDX3Y may compensate for loss of DDX3X in supporting NK-cell survival at the earliest stages of differentiation. We identify a crucial role for DDX3 helicases in the development and survival of NK cells that may have implications for the clinical use of DDX3 inhibitors as antiviral agents. R01 AI148080 R01 AR073228

During intracellular infection, T follicular helper (T FH ) and T helper 1 (T H 1) cells promote humoral and cell-mediated responses, respectively. Another subset, CD4-cytotoxic T lymphocytes (CD4-CTLs), eliminate infected cells via functions typically associated with CD8 + T cells. The mechanisms underlying differentiation of these populations are incompletely understood. Here, we identify the transcription factor Aiolos as a reciprocal regulator of T FH and CD4-CTL programming. We find that Aiolos deficiency results in downregulation of key T FH transcription factors, and consequently reduced T FH differentiation and antibody production, during influenza virus infection. Conversely, CD4-CTL programming is elevated, including enhanced Eomes and cytolytic molecule expression. We further demonstrate that Aiolos deficiency allows for enhanced IL-2 sensitivity and increased STAT5 association with CD4-CTL gene targets, including Eomes, effector molecules, and IL2Ra. Thus, our collective findings identify Aiolos as a pivotal regulator of CD4-CTL and T FH programming and highlight its potential as a target for manipulating CD4 + T cell responses.

The Ikaros zinc finger transcription factor Eos has been commonly implicated in regulatory T cells to promote their immunosuppressive functions. Paradoxically, a new role is emerging for Eos in promoting pro-inflammatory responses of conventional CD4 + T cells in the dysregulated setting of autoimmunity. Even so, the precise role of Eos in regulating the differentiation and function of healthy effector CD4 + T cell subsets remains unclear. Here, we find that Eos is a positive regulator of CD4 + T helper 2 (T H 2) cells—effector T cells implicated in the induction of allergic asthma. Using murine in vitro T H 2 cells and an in vivo house dust mite asthma model, we found that Eos-deficient T cells had reduced expression of key T H 2 transcription factors, effector cytokines, and differentiation receptors. Mechanistically, among various T H 2-polarizing pathways, the IL-2/STAT5 axis and its downstream T H 2 gene targets emerged as one of the most significantly downregulated networks in Eos deficiency. Using in vitro T H 2 cells and overexpression of Eos zinc-finger-domain mutants, we discovered that Eos forms a novel complex with and supports the tyrosine-phosphorylated signaling activity of STAT5. Overall, these data define a novel regulatory mechanism whereby Eos promotes IL-2/STAT5 activity to facilitate T H 2 differentiation.

PURPOSE OF REVIEW:Increases in ambient levels of air pollutants have been linked to lung inflammation and remodeling, processes that lead to the development and exacerbation of allergic asthma. Conventional research has focused on the role of CD4+ T helper 2 (TH2) cells in the pathogenesis of air pollution-induced asthma. However, much work in the past decade has uncovered an array of air pollution-induced non-TH2 immune mechanisms that contribute to allergic airway inflammation and disease.RECENT FINDINGS:In this article, we review current research demonstrating the connection between common air pollutants and their downstream effects on non-TH2 immune responses emerging as key players in asthma, including PRRs, ILCs, and non-TH2 T cell subsets. We also discuss the proposed mechanisms by which air pollution increases immune-mediated asthma risk, including pre-existing genetic risk, epigenetic alterations in immune cells, and perturbation of the composition and function of the lung and gut microbiomes. Together, these studies reveal the multifaceted impacts of various air pollutants on innate and adaptive immune functions via genetic, epigenetic, and microbiome-based mechanisms that facilitate the induction and worsening of asthma.

Effective immunity to influenza virus and other respiratory viruses requires the generation of CD4 + T cell subsets that coordinate multiple aspects of the immune response. These subsets include T follicular helper (T FH ) and T helper 1 (T H 1) cells, which promote humoral and cell-mediated responses, respectively. A third population, CD4 + cytotoxic T lymphocytes (CD4-CTLs) facilitates clearance of infection via mechanisms normally associated with CD8 + T cells. Here, we identify the transcription factor Aiolos as a regulator of T FH and CD4-CTL responses. We demonstrate that Aiolos deficiency compromises T FH differentiation and antibody production during influenza virus infection. Conversely, we find that CD4 + T cells acquire a cytotoxic-like program in the absence of Aiolos, including increased expression of the CTL-associated transcription factors Eomes and Blimp-1. We further show that while Aiolos positively regulates the T FH transcriptional regulators Zfp831, TCF-1 and Bcl-6, it also directly represses expression of IL-2Rα and IL-2/STAT5-driven expression of the cytotoxic gene program. Thus, our findings identify Aiolos as a pivotal regulator of T FH and CD4-CTL differentiation and highlight its potential as a target for manipulating CD4 + T cell humoral and cytotoxic responses.

Although CD4+ T helper 2 (TH2) cells normally defend against parasitic infection, they play a central role in the pathogenesis of allergic asthma. Therefore, understanding the molecular mechanisms governing TH2 differentiation and function is a top priority for improving asthma outcomes. One pathway of interest for controlling TH2-mediated disease is IL-2/STAT5 signaling, which is essential for TH2 polarization yet boasts a complex regulatory network that is incompletely understood. Here, we identify the Ikaros zinc finger (IkZF) transcription factor Eos as a candidate regulator of IL-2/STAT5 signaling underlying TH2 differentiation and function. To start, we show that Eos gene and protein expression is increased in TH2 cells relative to CD4+ T cell subsets that are negatively regulated by IL-2/STAT5 signaling, such as TH17 and TFH cells. Given these findings, we sought to define the functional effects of Eos in TH2 cells using in vitro and in vivo murine house dust mite asthma models. Intriguingly, Eos deficiency in vitro and in vivo results in reduced expression of key TH2 transcription factors (Gata3, Blimp-1), effector cytokines (IL-4, IL-13), and differentiation receptors (IL-2Rα, IL-2Rβ, IL-4Rα). Mechanistically, our data reveal that Eos interacts with and increases the activity of STAT5, suggesting that Eos enhances TH2 differentiation and effector function through direct STAT5 regulation. These findings in proinflammatory TH2 cells are of high significance, as Eos, to date, has largely been associated with the immunosuppressive functions of TREG cells. Taken together, our data reveal a novel mechanism by which Eos positively regulates IL-2/STAT5 signaling to promote TH2 differentiation and function. Work supported by grants from the NIH (R01AI134972) and The Ohio State University (Susan Huntington Dean’s Distinguished University Fellowship).

The X chromosome linked RNA helicase DDX3X plays numerous roles in regulation of cellular RNA metabolism and immune responses. Loss of function mutations in this helicase are linked to neurological disease and malignancies, including NK/T-cell lymphoma. To investigate how DDX3X contributes to development of NK cells, we generated mice with a conditional deletion Ddx3x in Ncr1-expressing cells. Female mice with homozygous deletion of NK cells exhibit a complete loss of NK cells. By comparison, the frequency of NK cells was significantly reduced in hemizygous male mice. The reduction in NK cells is associated with increased apoptosis in this lineage early during differentiation in the bone marrow. Our ongoing work is focused on elucidation of the mechanisms by which DDX3X regulates NK cell survival, differentiation, and malignant transformation, as well as on the capacity of DDX3Y to compensate for loss of DDX3X in hemizygous male mice. Our data identify a novel role for DDX3 RNA helicases in the development and survival of NK cells in mice.

Natural killer (NK) cell suppression of T cells is a key determinant of viral pathogenesis and vaccine efficacy. This process involves perforin-dependent elimination of activated CD4 T cells during the first three days of infection. Although this mechanism requires cell-cell contact, NK cells and T cells typically reside in different compartments of lymphoid tissues at steady state. Here, we show that NK-cell suppression of T cells is associated with a transient accumulation of NK cells within T cell-rich sites of the spleen during lymphocytic choriomeningitis virus infection. The chemokine receptor CXCR3 is required for relocation to T-cell zones and suppression of antiviral T cells. Accordingly, this NK-cell migration is mediated by type I interferon (IFN)-dependent promotion of CXCR3 ligand expression. In contrast, adenoviral vectors that weakly induce type I IFN and do not stimulate NK-cell inhibition of T cells also do not promote measurable redistribution of NK cells to T-cell zones. Provision of supplemental IFN could rescue NK-cell migration during adenoviral vector immunization. Thus, type I IFN and CXCR3 are critical for properly positioning NK cells to constrain antiviral T-cell responses. Development of strategies to curtail migration of NK cells between lymphoid compartments may enhance vaccine-elicited immune responses.

Abstract Natural killer (NK) cells dictate the pathogenic outcomes of infection via both direct killing of virus-infected cells and indirect immunoregulatory killing of antiviral T cells. The latter involves a perforin-dependent mechanism targeting activated CD4 T cells during the first three days of infection. Given that perforin-dependent killing involves cell-cell contact, we speculated that NK cells must re-locate proximal to recently activated T cells within the white pulp (WP) follicles of secondary lymphoid tissue during this critical temporal window of immunoregulatory activity. As expected, virus infection prompted a gradual accumulation of NK cells within T cell rich regions of splenic WP that peaked by the third day of infection. NK cells deficient in the chemokine receptor CXCR3 exhibited impaired localization to the WP and a markedly diminished capacity to suppress antiviral T-cell and germinal center B-cell responses. Our results reveal a critical role for CXCR3 in properly positioning NK cells to prune developing antiviral T cell responses, which potentially explains loss of tolerance and enhanced immune-mediated organ damage in absence of CXCR3. Strategies to curtail localization of NK cells in the WP during immunization may be an effective means to enhance vaccine-elicited immune responses.

Follicular helper T cells (TFH) are critical for vaccine and infection elicitation of long-lived humoral immunity, but exaggerated TFH responses can promote autoimmunity and other pathologies. It is unfortunate that no clinical interventions exist for the selective depletion of follicular T cells to alleviate these diseases. We engineered a chimeric antigen receptor (CAR) facilitating the specific targeting of cells with high expression levels of human programmed cell death protein 1 (PD-1), a cardinal feature of follicular T cells. CAR-expressing human natural killer (NK) cells robustly and discriminately eliminated PD-1high follicular human T cells in vitro and in a humanized mouse model of lupus-like disease while sparing B cells and other PD-1low T cell subsets, including regulatory T cells. These results establish a strategy for specific targeting of PD-1high T cells that can be advanced as a clinical tool for the selective depletion of pathogenic follicular T cells or other PD-1high target cells in certain disease states.

Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 is a novel and highly pathogenic coronavirus and is the causative agent of the coronavirus disease 2019 (COVID-19). The high morbidity and mortality associated with COVID-19 and the lack of an approved drug or vaccine for SARS-CoV-2 underscores the urgent need for developing effective antiviral therapies. Therapeutics that target essential viral proteins are effective at controlling virus replication and spread. Coronavirus Spike glycoproteins mediate viral entry and fusion with the host cell, and thus are essential for viral replication. To enter host cells, the Spike proteins of SARS-CoV-2 and related coronavirus, SARS-CoV, bind the host angiotensin-converting enzyme 2 (ACE2) receptor through their receptor binding domains (RBDs). Here, we rationally designed a panel of ACE2-derived peptides based on the RBD-ACE2 binding interfaces of SARS-CoV-2 and SARS-CoV. Using SARS-CoV-2 and SARS-CoV Spike-pseudotyped viruses, we found that a subset of peptides inhibits Spike-mediated infection with IC50 values in the low millimolar range. We identified two peptides that bound Spike RBD in affinity precipitation assays and inhibited infection with genuine SARS-CoV-2. Moreover, these peptides inhibited the replication of a common cold causing coronavirus, which also uses ACE2 as its entry receptor. Results from the infection experiments and modeling of the peptides with Spike RBD identified a 6-amino-acid (Glu37-Gln42) ACE2 motif that is important for SARS-CoV-2 inhibition. Our work demonstrates the feasibility of inhibiting SARS-CoV-2 with peptide-based inhibitors. These findings will allow for the successful development of engineered peptides and peptidomimetic-based compounds for the treatment of COVID-19.