PRMT5 Promotes T follicular helper Cell Differentiation and Germinal Center Responses during Influenza Virus Infection.

Protein arginine methyltransferases (PRMTs) modify diverse protein targets and regulate numerous cellular processes; yet, their contributions to individual effector T cell responses during infections are incompletely understood. In this study, we identify PRMT5 as a critical regulator of CD4+ T follicular helper cell (Tfh) responses during influenza virus infection in mice. Conditional PRMT5 deletion in murine T cells results in an almost complete ablation of both Tfh and T follicular regulatory populations and, consequently, reduced B cell activation and influenza-specific Ab production. Supporting a potential mechanism, we observe elevated surface expression of IL-2Rα on non-T regulatory effector PRMT5-deficient T cells. Notably, IL-2 signaling is known to negatively impact Tfh differentiation. Collectively, our findings identify PRMT5 as a prominent regulator of Tfh programming, with potential causal links to IL-2 signaling.

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.

TCDD and CH223191 Alter T Cell Balance but Fail to Induce Anti-Inflammatory Response in Adult Lupus Mice.

Aryl hydrocarbon receptor (AhR) responds to endogenous and exogenous ligands as a cytosolic receptor, transcription factor, and E3 ubiquitin ligase. Several studies support an anti-inflammatory effect of AhR activation. However, exposure to the AhR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) during early stages of development results in an autoimmune phenotype and exacerbates lupus. The effects of TCDD on lupus in adults with pre-existing autoimmunity have not been described. We present novel evidence that AhR stimulation by TCDD alters T cell responses but fails to impact lupus-like disease using an adult mouse model. Interestingly, AhR antagonist CH223191 also changed T cell balance in our model. We next developed a conceptual framework for identifying cellular and molecular factors that contribute to physiological outcomes in lupus and created models that describe cytokine dynamics that were fed into a system of differential equations to predict the kinetics of T follicular helper (Tfh) and regulatory T (Treg) cell populations. The model predicted that Tfh cells expanded to larger values following TCDD exposure compared with vehicle and CH223191. Following the initial elevation, both Tfh and Treg cell populations continuously decayed over time. A function based on the ratio of predicted Treg/Tfh cells showed that Treg cells exceed Tfh cells in all groups, with TCDD and CH223191 showing lower Treg/Tfh cell ratios than the vehicle and that the ratio is relatively constant over time. We conclude that AhR ligands did not induce an anti-inflammatory response to attenuate autoimmunity in adult lupus mice. This study challenges the dogma that TCDD supports an immunosuppressive phenotype.

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.

Prime-Pull Immunization of Mice with a BcfA-Adjuvanted Vaccine Elicits Sustained Mucosal Immunity That Prevents SARS-CoV-2 Infection and Pathology.

Vaccines against SARS-CoV-2 that induce mucosal immunity capable of preventing infection and disease remain urgently needed. In this study, we demonstrate the efficacy of Bordetella colonization factor A (BcfA), a novel bacteria-derived protein adjuvant, in SARS-CoV-2 spike-based prime-pull immunizations. We show that i.m. priming of mice with an aluminum hydroxide- and BcfA-adjuvanted spike subunit vaccine, followed by a BcfA-adjuvanted mucosal booster, generated Th17-polarized CD4+ tissue-resident memory T cells and neutralizing Abs. Immunization with this heterologous vaccine prevented weight loss following challenge with mouse-adapted SARS-CoV-2 (MA10) and reduced viral replication in the respiratory tract. Histopathology showed a strong leukocyte and polymorphonuclear cell infiltrate without epithelial damage in mice immunized with BcfA-containing vaccines. Importantly, neutralizing Abs and tissue-resident memory T cells were maintained until 3 mo postbooster. Viral load in the nose of mice challenged with the MA10 virus at this time point was significantly reduced compared with naive challenged mice and mice immunized with an aluminum hydroxide-adjuvanted vaccine. We show that vaccines adjuvanted with alum and BcfA, delivered through a heterologous prime-pull regimen, provide sustained protection against SARS-CoV-2 infection.

Aiolos represses CD4+ T cell cytotoxic programming via reciprocal regulation of TFH transcription factors and IL-2 sensitivity.

During intracellular infection, T follicular helper (TFH) and T helper 1 (TH1) 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 TFH and CD4-CTL programming. We find that Aiolos deficiency results in downregulation of key TFH transcription factors, and consequently reduced TFH 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 TFH programming and highlight its potential as a target for manipulating CD4+ T cell responses.

SUSD2 suppresses CD8+ T cell antitumor immunity by targeting IL-2 receptor signaling.

Dysfunctional CD8+ T cells, which have defective production of antitumor effectors, represent a major mediator of immunosuppression in the tumor microenvironment. Here, we show that SUSD2 is a negative regulator of CD8+ T cell antitumor function. Susd2-/- effector CD8+ T cells showed enhanced production of antitumor molecules, which consequently blunted tumor growth in multiple syngeneic mouse tumor models. Through a quantitative mass spectrometry assay, we found that SUSD2 interacted with interleukin (IL)-2 receptor α through sushi domain-dependent protein interactions and that this interaction suppressed the binding of IL-2, an essential cytokine for the effector functions of CD8+ T cells, to IL-2 receptor α. SUSD2 was not expressed on regulatory CD4+ T cells and did not affect the inhibitory function of these cells. Adoptive transfer of Susd2-/- chimeric antigen receptor T cells induced a robust antitumor response in mice, highlighting the potential of SUSD2 as an immunotherapy target for cancer.

Evaluation of early innate and adaptive immune responses to the TB vaccine Mycobacterium bovis BCG and vaccine candidate BCGΔBCG1419c.

The vaccine Mycobacterium bovis Bacillus Calmette-Guérin (BCG) elicits an immune response that is protective against certain forms of tuberculosis (TB); however, because BCG efficacy is limited it is important to identify alternative TB vaccine candidates. Recently, the BCG deletion mutant and vaccine candidate BCGΔBCG1419c was demonstrated to survive longer in intravenously infected BALB/c mice due to enhanced biofilm formation, and better protected both BALB/c and C57BL/6 mice against TB-induced lung pathology during chronic stages of infection, relative to BCG controls. BCGΔBCG1419c-elicited protection also associated with lower levels of proinflammatory cytokines (i.e. IL6, TNFα) at the site of infection in C57BL/6 mice. Given the distinct immune profiles of BCG- and BCGΔBCG1419c-immunized mice during chronic TB, we set out to determine if there are early immunological events which distinguish these two groups, using multi-dimensional flow cytometric analysis of the lungs and other tissues soon after immunization. Our results demonstrate a number of innate and adaptive response differences between BCG- and BCGΔBCG1419c-immunized mice which are consistent with the latter being longer lasting and potentially less inflammatory, including lower frequencies of exhausted CD4+ T helper (TH) cells and higher frequencies of IL10-producing T cells, respectively. These studies suggest the use of BCGΔBCG1419c may be advantageous as an alternative TB vaccine candidate.

Pericyte Progenitor Coupling to the Emerging Endothelium During Vasculogenesis via Connexin 43.

BACKGROUND: Vascular pericytes stabilize blood vessels and contribute to their maturation, while playing other key roles in microvascular function. Nevertheless, relatively little is known about involvement of their precursors in the earliest stages of vascular development, specifically during vasculogenesis. METHODS: We combined high-power, time-lapse imaging with transcriptional profiling of emerging pericytes and endothelial cells in reporter mouse and cell lines. We also analyzed conditional transgenic animals deficient in Cx43/Gja1 (connexin 43/gap junction alpha-1) expression within Ng2+ cells. RESULTS: A subset of Ng2-DsRed+ cells, likely pericyte/mural cell precursors, arose alongside endothelial cell differentiation and organization and physically engaged vasculogenic endothelium in vivo and in vitro. We found no overlap between this population of differentiating pericyte/mural progenitors and other lineages including hemangiogenic and neuronal/glial cell types. We also observed cell-cell coupling and identified Cx43-based gap junctions contributing to pericyte-endothelial cell precursor communication during vascular assembly. Genetic loss of Cx43/Gja1 in Ng2+ pericyte progenitors compromised embryonic blood vessel formation in a subset of animals, while surviving mutants displayed little-to-no vessel abnormalities, suggesting a resilience to Cx43/Gja1 loss in Ng2+ cells or potential compensation by additional connexin isoforms. CONCLUSIONS: Together, our data suggest that a distinct pericyte lineage emerges alongside vasculogenesis and directly communicates with the nascent endothelium via Cx43 during early vessel formation. Cx43/Gja1 loss in pericyte/mural cell progenitors can induce embryonic vessel dysmorphogenesis, but alternate connexin isoforms may be able to compensate. These data provide insight that may reshape the current framework of vascular development and may also inform tissue revascularization/vascularization strategies.

Making memories with Bcl-6.

For over a decade, mutual antagonism between the transcriptional repressors Bcl-6 and Blimp-1 has been appreciated as a key mechanistic determinant of lymphoid differentiation programs. Now, in this issue of JEM, Ciucci et al. (2021. J. Exp. Med.https://doi.org/10.1084/jem.20202343) demonstrate that this relationship is "central" to the generation of T cell memory.

Established and emergent roles for Ikaros transcription factors in lymphoid cell development and function.

Ikaros zinc finger transcription factors are important regulators of the gene programs underlying the development of hematopoietic cell lineages. The family consists of five members: Ikaros, Helios, Aiolos, Eos, and Pegasus, which engage in both homo- and heterotypic intrafamilial interactions to exert diverse functional effects. Pioneering studies focused on the role of these factors in early lymphoid development, as their absence resulted in severe defects in lymphocyte populations. More recent work has now begun to define nuanced, stage-specific roles for Ikaros family members in the differentiation and function of mature T, B, and innate lymphoid cell populations including natural killer (NK) cells. The precise transcriptional mechanisms by which these factors function, both independently and collaboratively, is an area of active investigation. However, several key themes appear to be emerging regarding the pathways influenced by Ikaros family members, including the end-to-end regulation of cytokine signaling. Here, we review roles for Ikaros factors in lymphoid cell development, differentiation, and function, including a discussion of the current understanding of the transcriptional mechanisms they employ and considerations for the future study of this important transcription factor family.

Obesity-Driven Deficiencies of Specialized Pro-resolving Mediators May Drive Adverse Outcomes During SARS-CoV-2 Infection.

Obesity is a major independent risk factor for increased morbidity and mortality upon infection with Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV-2), which is responsible for the current coronavirus disease pandemic (COVID-19). Therefore, there is a critical need to identify underlying metabolic factors associated with obesity that could be contributing toward increased susceptibility to SARS-CoV-2 in this vulnerable population. Here, we focus on the critical role of potent endogenous lipid metabolites known as specialized pro-resolving mediators (SPMs) that are synthesized from polyunsaturated fatty acids. SPMs are generated during the transition of inflammation to resolution and have a vital role in directing damaged tissues to homeostasis; furthermore, SPMs display anti-viral activity in the context of influenza infection without being immunosuppressive. We cover evidence from rodent and human studies to show that obesity, and its co-morbidities, induce a signature of SPM deficiency across immunometabolic tissues. We further discuss how the effects of obesity upon SARS-CoV-2 infection are likely exacerbated with environmental exposures that promote chronic pulmonary inflammation and augment SPM deficits. Finally, we highlight potential approaches to overcome the loss of SPMs using dietary and pharmacological interventions. Collectively, this mini-review underscores the need for mechanistic studies on how SPM deficiencies driven by obesity and environmental exposures may exacerbate the response to SARS-CoV-2.

Dynamic Roles for IL-2-STAT5 Signaling in Effector and Regulatory CD4+ T Cell Populations.

CD4+ Th cells are responsible for orchestrating diverse, pathogen-specific immune responses through their differentiation into a number of subsets, including TH1, TH2, TH9, T follicular helper, T follicular regulatory, and regulatory T cells. The differentiation of each subset is guided by distinct regulatory requirements, including those derived from extracellular cytokine signals. IL-2 has emerged as a critical immunomodulatory cytokine that both positively and negatively affects the differentiation of individual Th cell subsets. IL-2 signals are propagated, in part, via activation of STAT5, which functions as a key regulator of CD4+ T cell gene programs. In this review, we discuss current understanding of the mechanisms that allow IL-2-STAT5 signaling to exert divergent effects across CD4+ T cell subsets and highlight specific roles for this pathway in the regulation of individual Th cell differentiation programs.

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.

Inhibition of IL-2 responsiveness by IL-6 is required for the generation of GC-TFH cells.

Sustained T cell receptor (TCR) stimulation is required for maintaining germinal center T follicular helper (GC-TFH) cells. Paradoxically, TCR activation induces interleukin-2 receptor (IL-2R) expression and IL-2 production, thereby initiating a feedback loop of IL-2 signaling that normally inhibits TFH cells. It is unclear how GC-TFH cells can receive prolonged TCR signaling without succumbing to the detrimental effects of IL-2. Using an influenza infection model, we show here that GC-TFH cells secreted large amounts of IL-2 but responded poorly to it. To maintain their IL-2 hyporesponsiveness, GC-TFH cells required intrinsic IL-6 signaling. Mechanistically, we found that IL-6 inhibited up-regulation of IL-2Rß (CD122) by preventing association of STAT5 with the Il2rb locus, thus allowing GC-TFH cells to receive sustained TCR signaling and produce IL-2 without initiating a TCR/IL-2 inhibitory feedback loop. Collectively, our results identify a regulatory mechanism that controls the generation of GC-TFH cells.

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.

High-frequency irreversible electroporation is an effective tumor ablation strategy that induces immunologic cell death and promotes systemic anti-tumor immunity.

BACKGROUND: Despite promising treatments for breast cancer, mortality rates remain high and treatments for metastatic disease are limited. High-frequency irreversible electroporation (H-FIRE) is a novel tumor ablation technique that utilizes high-frequency bipolar electric pulses to destabilize cancer cell membranes and induce cell death. However, there is currently a paucity of data pertaining to immune system activation following H-FIRE and other electroporation based tumor ablation techniques. METHODS: Here, we utilized the mouse 4T1 mammary tumor model to evaluate H-FIRE treatment parameters on cancer progression and immune system activation in vitro and in vivo. FINDINGS: H-FIRE effectively ablates the primary tumor and induces a pro-inflammatory shift in the tumor microenvironment. We further show that local treatment with H-FIRE significantly reduces 4T1 metastases. H-FIRE kills 4T1 cells through non-thermal mechanisms associated with necrosis and pyroptosis resulting in damage associated molecular pattern signaling in vitro and in vivo. Our data indicate that the level of tumor ablation correlates with increased activation of cellular immunity. Likewise, we show that the decrease in metastatic lesions is dependent on the intact immune system and H-FIRE generates 4T1 neoantigens that engage the adaptive immune system to significantly attenuate tumor progression. INTERPRETATION: Cell death and tumor ablation following H-FIRE treatment activates the local innate immune system, which shifts the tumor microenvironment from an anti-inflammatory state to a pro-inflammatory state. The non-thermal damage to the cancer cells and increased innate immune system stimulation improves antigen presentation, resulting in the engagement of the adaptive immune system and improved systemic anti-tumor immunity.

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.

Systemic Listeria monocytogenes Infection as a Model to Study T Helper Cell Immune Responses.

Listeria monocytogenes, a Gram-positive facultative intracellular pathogen, has been widely used as a model for studying the immune response. Here, we describe a protocol for the systemic infection of mice with L. monocytogenes, followed by isolation of lymphocytes from spleens and lymph nodes. We also include details on how to culture and store L. monocytogenes, as well as the specifics for fluorescence-activated cell sorting (FACS) for CD4+ cells in response to the systemic infection. This protocol can be adapted by changing the dosage of L. monocytogenes for a more or less aggressive infection and/or sorting for other immune cell subtypes of interest.

Additive manufacturing of three-dimensional (3D) microfluidic-based microelectromechanical systems (MEMS) for acoustofluidic applications.

Three-dimensional (3D) printing now enables the fabrication of 3D structural electronics and microfluidics. Further, conventional subtractive manufacturing processes for microelectromechanical systems (MEMS) relatively limit device structure to two dimensions and require post-processing steps for interface with microfluidics. Thus, the objective of this work is to create an additive manufacturing approach for fabrication of 3D microfluidic-based MEMS devices that enables 3D configurations of electromechanical systems and simultaneous integration of microfluidics. Here, we demonstrate the ability to fabricate microfluidic-based acoustofluidic devices that contain orthogonal out-of-plane piezoelectric sensors and actuators using additive manufacturing. The devices were fabricated using a microextrusion 3D printing system that contained integrated pick-and-place functionality. Additively assembled materials and components included 3D printed epoxy, polydimethylsiloxane (PDMS), silver nanoparticles, and eutectic gallium-indium as well as robotically embedded piezoelectric chips (lead zirconate titanate (PZT)). Electrical impedance spectroscopy and finite element modeling studies showed the embedded PZT chips exhibited multiple resonant modes of varying mode shape over the 0-20 MHz frequency range. Flow visualization studies using neutrally buoyant particles (diameter = 0.8-70 µm) confirmed the 3D printed devices generated bulk acoustic waves (BAWs) capable of size-selective manipulation, trapping, and separation of suspended particles in droplets and microchannels. Flow visualization studies in a continuous flow format showed suspended particles could be moved toward or away from the walls of microfluidic channels based on selective actuation of in-plane or out-of-plane PZT chips. This work suggests additive manufacturing potentially provides new opportunities for the design and fabrication of acoustofluidic and microfluidic devices.