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.

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.

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.

Selective Histone Deacetylase 6 Inhibition Normalizes B Cell Activation and Germinal Center Formation in a Model of Systemic Lupus Erythematosus.

Autoantibody production by plasma cells (PCs) plays a pivotal role in the pathogenesis of systemic lupus erythematosus (SLE). The molecular pathways by which B cells become pathogenic PC secreting autoantibodies in SLE are incompletely characterized. Histone deactylase 6 (HDAC6) is a unique cytoplasmic HDAC that modifies the interaction of a number of tubulin- associated proteins; inhibition of HDAC6 has been shown to be beneficial in murine models of SLE, but the downstream pathways accounting for the therapeutic benefit have not been clearly delineated. In the current study, we sought to determine whether selective HDAC6 inhibition would abrogate abnormal B cell activation in SLE. We treated NZB/W lupus mice with the selective HDAC6 inhibitor, ACY-738, for 4 weeks beginning at 20 weeks-of age. After only 4 weeks of treatment, manifestation of lupus nephritis (LN) were greatly reduced in these animals. We then used RNAseq to determine the genomic signatures of splenocytes from treated and untreated mice and applied computational cellular and pathway analysis to reveal multiple signaling events associated with B cell activation and differentiation in SLE that were modulated by HDAC6 inhibition. PC development was abrogated and germinal center (GC) formation was greatly reduced. When the HDAC6 inhibitor-treated lupus mouse gene signatures were compared to human lupus patient gene signatures, the results showed numerous immune, and inflammatory pathways increased in active human lupus were significantly decreased in the HDAC6 inhibitor treated animals. Pathway analysis suggested alterations in cellular metabolism might contribute to the normalization of lupus mouse spleen genomic signatures, and this was confirmed by direct measurement of the impact of the HDAC6 inhibitor on metabolic activities of murine spleen cells. Taken together, these studies show HDAC6 inhibition decreases B cell activation signaling pathways and reduces PC differentiation in SLE and suggest that a critical event might be modulation of cellular metabolism.

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.

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.

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.

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.

Control of lupus nephritis by changes of gut microbiota.

BACKGROUND: Systemic lupus erythematosus, characterized by persistent inflammation, is a complex autoimmune disorder with no known cure. Immunosuppressants used in treatment put patients at a higher risk of infections. New knowledge of disease modulators, such as symbiotic bacteria, can enable fine-tuning of parts of the immune system, rather than suppressing it altogether. RESULTS: Dysbiosis of gut microbiota promotes autoimmune disorders that damage extraintestinal organs. Here we report a role of gut microbiota in the pathogenesis of renal dysfunction in lupus. Using a classical model of lupus nephritis, MRL/lpr, we found a marked depletion of Lactobacillales in the gut microbiota. Increasing Lactobacillales in the gut improved renal function of these mice and prolonged their survival. We used a mixture of 5 Lactobacillus strains (Lactobacillus oris, Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillus johnsonii, and Lactobacillus gasseri), but L. reuteri and an uncultured Lactobacillus sp. accounted for most of the observed effects. Further studies revealed that MRL/lpr mice possessed a "leaky" gut, which was reversed by increased Lactobacillus colonization. Lactobacillus treatment contributed to an anti-inflammatory environment by decreasing IL-6 and increasing IL-10 production in the gut. In the circulation, Lactobacillus treatment increased IL-10 and decreased IgG2a that is considered to be a major immune deposit in the kidney of MRL/lpr mice. Inside the kidney, Lactobacillus treatment also skewed the Treg-Th17 balance towards a Treg phenotype. These beneficial effects were present in female and castrated male mice, but not in intact males, suggesting that the gut microbiota controls lupus nephritis in a sex hormone-dependent manner. CONCLUSIONS: This work demonstrates essential mechanisms on how changes of the gut microbiota regulate lupus-associated immune responses in mice. Future studies are warranted to determine if these results can be replicated in human subjects.

T follicular helper cell programming by cytokine-mediated events.

CD4+ T cells, or T helper cells, are critical mediators and coordinators of adaptive immunity. Unique effector T helper cell populations have been identified that perform distinct functions in response to pathogenic infection. The T follicular helper (Tfh) cells are one such subset, which has been identified as the primary T-cell population responsible for interacting with B cells to promote effective antibody-mediated immune responses. Since their initial description at the turn of the century, and subsequent classification as a distinct T helper cell subset, there has been substantial interest in elucidating the regulatory mechanisms that govern Tfh cell formation. The collective insight from this body of work has demonstrated that Tfh cell differentiation is a complex and multistage process regulated by a litany of cell-intrinsic and cell-extrinsic factors. As with the development of the other recognized T helper cell subsets, specific cytokines exercise prominent roles in both the positive and negative regulation of Tfh cell development. However, the exact composition of, and stage-specific requirements for, these environmental factors in the governance of Tfh cell differentiation remain incompletely understood. In this review, we summarize what is known regarding the role of cytokines in both the promotion and inhibition of Tfh cell differentiation and function.

IL-2, IL-7, and IL-15: Multistage regulators of CD4(+) T helper cell differentiation.

Cytokines represent a class of environmental factors that are critical drivers of immune cell development. Cytokines of the common gamma-chain family, including interleukin (IL)-2, IL-7, and IL-15, have been the subject of intense experimental scrutiny and have well-defined roles as regulators of diverse immune cell types including CD4(+) T helper cells. Because of their pleiotropic effects on T-cell development and function, researchers and clinicians have attempted to harness the capabilities of these cytokines for therapeutic benefit. In this review, we summarize the recent progress in our understanding of the molecular mechanisms underlying the effects of these cytokines on CD4(+) T cell development and briefly discuss how these immunomodulatory cytokines are being used in efforts to treat human disease.

IL-7 signalling represses Bcl-6 and the TFH gene program.

The transcriptional repressor Bcl-6 is linked to the development of both CD4(+) T follicular helper (TFH) and central memory T (TCM) cells. Here, we demonstrate that in response to decreased IL-2 signalling, T helper 1 (TH1) cells upregulate Bcl-6 and co-initiate TFH- and TCM-like gene programs, including expression of the cytokine receptors IL-6Rα and IL-7R. Exposure of this potentially bi-potent cell population to IL-6 favours the TFH gene program, whereas IL-7 signalling represses TFH-associated genes including Bcl6 and Cxcr5, but not the TCM-related genes Klf2 and Sell. Mechanistically, IL-7-dependent activation of STAT5 contributes to Bcl-6 repression. Importantly, antigen-specific IL-6Rα(+)IL-7R(+) CD4(+) T cells emerge from the effector population at late time points post influenza infection. These data support a novel role for IL-7 in the repression of the TFH gene program and evoke a divergent regulatory mechanism by which post-effector TH1 cells may contribute to long-term cell-mediated and humoral immunity.