Interferon-γ production by Tfh cells is required for CXCR3+ pre-memory B cell differentiation and subsequent lung-resident memory B cell responses.

Lung-resident memory B cells (lung-BRMs) differentiate into plasma cells after reinfection, providing enhanced pulmonary protection. Here, we investigated the determinants of lung-BRM differentiation upon influenza infection. Kinetic analyses revealed that influenza nucleoprotein (NP)-specific BRMs preferentially differentiated early after infection and required T follicular helper (Tfh) cell help. BRM differentiation temporally coincided with transient interferon (IFN)-γ production by Tfh cells. Depletion of IFN-γ in Tfh cells prevented lung-BRM differentiation and impaired protection against heterosubtypic infection. IFN-γ was required for expression of the transcription factor T-bet by germinal center (GC) B cells, which promoted differentiation of a CXCR3+ GC B cell subset that were precursors of lung-BRMs and CXCR3+ memory B cells in the mediastinal lymph node. Absence of IFN-γ signaling or T-bet in GC B cells prevented CXCR3+ pre-memory precursor development and hampered CXCR3+ memory B cell differentiation and subsequent lung-BRM responses. Thus, Tfh-cell-derived IFN-γ is critical for lung-BRM development and pulmonary immunity, with implications for vaccination strategies targeting BRMs.

IL-6 prevents Th2 cell polarization by promoting SOCS3-dependent suppression of IL-2 signaling.

Defective interleukin-6 (IL-6) signaling has been associated with Th2 bias and elevated IgE levels. However, the underlying mechanism by which IL-6 prevents the development of Th2-driven diseases remains unknown. Using a model of house dust mite (HDM)-induced Th2 cell differentiation and allergic airway inflammation, we showed that IL-6 signaling in allergen-specific T cells was required to prevent Th2 cell differentiation and the subsequent IgE response and allergic inflammation. Th2 cell lineage commitment required strong sustained IL-2 signaling. We found that IL-6 turned off IL-2 signaling during early T-cell activation and thus inhibited Th2 priming. Mechanistically, IL-6-driven inhibition of IL-2 signaling in responding T cells was mediated by upregulation of Suppressor Of Cytokine Signaling 3 (SOCS3). This mechanism could be mimicked by pharmacological Janus Kinase-1 (JAK1) inhibition. Collectively, our results identify an unrecognized mechanism that prevents the development of unwanted Th2 cell responses and associated diseases and outline potential preventive interventions.

Assessment of the Impact of Cytokines on TFH, TREG, and TFR Cell Populations After Influenza Infection.

Within the last several years, great strides have been made in understanding the molecular and cellular mechanisms that control the generation of T follicular helper (TFH), T regulatory (TREG), and T follicular regulatory (TFR) cells. As a result, it is now clear that cytokines play a critical role in regulating the development and function of these CD4+ T cell subsets. One of the critical limitations when studying the effect of individual cytokines in these populations is differentiating between the intrinsic and extrinsic effects of these cytokines in vivo. Here we describe how to utilize mixed bone marrow chimeras in combination with MHC class II tetramers to characterize the direct role played by cytokines on controlling the development, function, and maintenance of TFH, TREG, and TFR cells in vivo.

GM-CSF production by non-classical monocytes controls antagonistic LPS-driven functions in allergic inflammation.

Lipopolysaccharide (LPS) can either promote or prevent T helper 2 (Th2) cell allergic responses. However, the underlying mechanism remains unknown. We show here that LPS activity switches from pro-pathogenic to protective depending on the production of granulocyte-macrophage colony-stimulating factor (GM-CSF) by non-classical monocytes. In the absence of GM-CSF, LPS can favor pathogenic Th2 cell responses by supporting the trafficking of lung-migratory dendritic cells (mDC2s) into the lung-draining lymph node. However, when non-classical monocytes produce GM-CSF, LPS and GM-CSF synergize to differentiate monocyte-derived DCs from classical Ly6Chi monocytes that instruct mDC2s for Th2 cell suppression. Importantly, only allergens with cysteine protease activity trigger GM-CSF production by non-classical monocytes. Hence, the therapeutic effect of LPS is restricted to allergens with this enzymatic activity. Treatment with GM-CSF, however, restores the protective effects of LPS. Thus, GM-CSF produced by non-classical monocytes acts as a rheostat that fine-tunes the pathogenic and therapeutic functions of LPS.

Lung dendritic cells migrate to the spleen to prime long-lived TCF1hi memory CD8+ T cell precursors after influenza infection.

CD8+ T cell responses to pulmonary challenges are primed by lung migratory dendritic cells (mDCs), which capture antigens in the lungs and migrate to the lung-draining mediastinal lymph node (med-LN) to activate T cells. The lungs and the spleen are not connected by the lymphatic vasculature. Thus, the current paradigm suggests that, in response to respiratory virus infections that are restricted to the respiratory tract, priming of T cell responses by lung mDCs takes place entirely in the med-LN. Our results challenge this "LN-centric" paradigm by demonstrating that, during influenza virus infection, lung mDCs egress the med-LN and traffic to the spleen, where they prime influenza-specific CD8+ T cells. CD8+ T cells primed in the spleen are transcriptionally distinct and have enhanced ability to differentiate into long-lived memory cells compared with med-LN­primed counterparts. Thus, our data identify a lung mDC trafficking pathway that connects the lungs with the spleen.

The Potential of Harnessing IL-2-Mediated Immunosuppression to Prevent Pathogenic B Cell Responses.

Immunosuppressive drugs can partially control Antibody (Ab)-dependent pathology. However, these therapeutic regimens must be maintained for the patient's lifetime, which is often associated with severe side effects. As research advances, our understanding of the cellular and molecular mechanisms underlying the development and maintenance of auto-reactive B cell responses has significantly advanced. As a result, novel immunotherapies aimed to restore immune tolerance and prevent disease progression in autoimmune patients are underway. In this regard, encouraging results from clinical and preclinical studies demonstrate that subcutaneous administration of low-doses of recombinant Interleukin-2 (r-IL2) has potent immunosuppressive effects in patients with autoimmune pathologies. Although the exact mechanism by which IL-2 induces immunosuppression remains unclear, the clinical benefits of the current IL-2-based immunotherapies are attributed to its effect on bolstering T regulatory (Treg) cells, which are known to suppress overactive immune responses. In addition to Tregs, however, rIL-2 also directly prevent the T follicular helper cells (Tfh), T helper 17 cells (Th17), and Double Negative (DN) T cell responses, which play critical roles in the development of autoimmune disorders and have the ability to help pathogenic B cells. Here we discuss the broader effects of rIL-2 immunotherapy and the potential of combining rIL-2 with other cytokine-based therapies to more efficiently target Tfh cells, Th17, and DN T cells and subsequently inhibit auto-antibody (ab) production in autoimmune patients.

Modulating Th2 Cell Immunity for the Treatment of Asthma.

It is estimated that more than 339 million people worldwide suffer from asthma. The leading cause of asthma development is the breakdown of immune tolerance to inhaled allergens, prompting the immune system's aberrant activation. During the early phase, also known as the sensitization phase, allergen-specific T cells are activated and become central players in orchestrating the subsequent development of allergic asthma following secondary exposure to the same allergens. It is well-established that allergen-specific T helper 2 (Th2) cells play central roles in developing allergic asthma. As such, 80% of children and 60% of adult asthma cases are linked to an unwarranted Th2 cell response against respiratory allergens. Thus, targeting essential components of Th2-type inflammation using neutralizing antibodies against key Th2 modulators has recently become an attractive option for asthmatic patients with moderate to severe symptoms. In addition to directly targeting Th2 mediators, allergen immunotherapy, also known as desensitization, is focused on redirecting the allergen-specific T cells response from a Th2-type profile to a tolerogenic one. This review highlights the current understanding of the heterogeneity of the Th2 cell compartment, their contribution to allergen-induced airway inflammation, and the therapies targeting the Th2 cell pathway in asthma. Further, we discuss available new leads for successful targeting pulmonary Th2 cell responses for future therapeutics.

Characterization and regulation of osteoclast precursors following chronic Porphyromonas gingivalis infection.

Bone destruction in inflammatory osteolytic diseases including periodontitis is related to excessive activity of osteoclasts (OC), which originate from precursor cells of the myeloid lineage, termed osteoclast precursors (OCP). In contrast to ample knowledge that we currently have on mature OC, little is known about OCP and their regulation during bacterial infection. Therefore, this study aimed to identify and characterize OCP following chronic infection with a periodontal bacteria Porphyromonas gingivalis (Pg). We used a micro-osmotic pump to continually release Pg subcutaneously in a murine model. Two weeks after Pg infection, the frequency of CD11b+c-fms+Ly6Chi population is significantly elevated within the bone marrow, spleen and peripheral blood. In vitro and in vivo studies identified these cells as the OCP-containing population and Pg infection significantly enhanced the osteoclastogenic activity of these cells. Furthermore, mRNA sequencing analysis indicated a unique gene and pathway profile in CD11b+c-fms+Ly6Chi population following Pg infection, with changes in genes and pathways related to OC differentiation, cell proliferation and apoptosis, inflammatory response, phagocytosis and immunity, as well as antigen processing and presentation. Moreover, using IL-6 knockout mice, we found that IL-6 is important for Pg-induced accumulation of CD11b+c-fms+Ly6Chi population from the bone marrow and periphery. Our results provide new insights into the characterization and regulation of OCP following a chronic bacterial infection. This knowledge is relevant to the understanding of the pathogenesis of bacteria-induced bone loss, and to the identification of potential therapeutic targets of bone loss diseases.

Frontline Science: Characterization and regulation of osteoclast precursors following chronic Porphyromonas gingivalis infection.

Bone destruction in inflammatory osteolytic diseases including periodontitis is related to excessive activity of osteoclasts (OC), which originate from precursor cells of the myeloid lineage, termed osteoclast precursors (OCP). In contrast to ample knowledge that we currently have on mature OC, little is known about OCP and their regulation during bacterial infection. Therefore, this study aimed to identify and characterize OCP following chronic infection with a periodontal bacteria Porphyromonas gingivalis (Pg). We used a microosmotic pump to continually release Pg subcutaneously in a murine model. Two weeks after Pg infection, the frequency of CD11b+c-fms+Ly6Chi population is significantly elevated within the bone marrow, spleen, and peripheral blood. In vitro and in vivo studies identified these cells as the OCP-containing population and Pg infection significantly enhanced the osteoclastogenic activity of these cells. Furthermore, mRNA sequencing analysis indicated a unique gene and pathway profile in CD11b+c-fms+Ly6Chi population following Pg infection, with changes in genes and pathways related to OC differentiation, cell proliferation and apoptosis, inflammatory response, phagocytosis, and immunity, as well as antigen processing and presentation. Moreover, using IL-6 knockout mice, we found that IL-6 is important for Pg-induced accumulation of CD11b+c-fms+Ly6Chi population from the bone marrow and periphery. Our results provide new insight into the characterization and regulation of OCP following a chronic bacterial infection. This knowledge is relevant to the understanding of the pathogenesis of bacteria-induced bone loss, and to the identification of potential therapeutic targets of bone loss diseases.

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.

Mechanisms of action of low-dose IL-2 restoration therapies in SLE.

Interleukin-2 (IL-2) shortage is a hallmark of Systemic Lupus Erythematosus (SLE). Importantly, clinical and preclinical studies demonstrate the potential clinical benefits of IL-2-based restoration therapies for the treatment of SLE. Here we discuss the immunological consequences of IL-2 deficiency in SLE patients and the mechanisms underlying the therapeutic effects of low-dose IL-2 regimens.

T-bet Transcription Factor Promotes Antibody-Secreting Cell Differentiation by Limiting the Inflammatory Effects of IFN-γ on B Cells.

Although viral infections elicit robust interferon-γ (IFN-γ) and long-lived antibody-secreting cell (ASC) responses, the roles for IFN-γ and IFN-γ-induced transcription factors (TFs) in ASC development are unclear. We showed that B cell intrinsic expression of IFN-γR and the IFN-γ-induced TF T-bet were required for T-helper 1 cell-induced differentiation of B cells into ASCs. IFN-γR signaling induced Blimp1 expression in B cells but also initiated an inflammatory gene program that, if not restrained, prevented ASC formation. T-bet did not affect Blimp1 upregulation in IFN-γ-activated B cells but instead regulated chromatin accessibility within the Ifng and Ifngr2 loci and repressed the IFN-γ-induced inflammatory gene program. Consistent with this, B cell intrinsic T-bet was required for formation of long-lived ASCs and secondary ASCs following viral, but not nematode, infection. Therefore, T-bet facilitates differentiation of IFN-γ-activated inflammatory effector B cells into ASCs in the setting of IFN-γ-, but not IL-4-, induced inflammatory responses.

Dysregulation of T Follicular Helper Cells in Lupus.

Although multiple and overlapping mechanisms are ultimately responsible for the immunopathology observed in patients with systemic lupus erythematosus, autoreactive Abs secreted by autoreactive plasma cells (PCs) are considered to play a critical role in disease progression and immunopathology. Given that PCs derive from the germinal centers (GC), long-term dysregulated GC reactions are often associated with the development of spontaneous autoantibody responses and immunopathology in systemic lupus erythematosus patients. In this review, we summarize the emerging evidence concerning the roles of T follicular helper cells in regulating pathogenic GC and autoreactive PC responses in lupus.

Impaired Tumor-Necrosis-Factor-α-driven Dendritic Cell Activation Limits Lipopolysaccharide-Induced Protection from Allergic Inflammation in Infants.

Infants have a higher risk of developing allergic asthma than adults. However, the underlying mechanism remains unknown. We show here that sensitization of mice with house-dust mites (HDMs) in the presence of low-dose lipopolysaccharide (LPS) prevented T helper 2 (Th2) cell allergic responses in adult, but not infant, mice. Mechanistically, adult CD11b+ migratory dendritic cells (mDCs) upregulated the transcription factor T-bet in response to tumor necrosis factor-α (TNF-α), which was rapidly induced after HDM + LPS sensitization. Consequently, adult CD11b+ mDCs produced interleukin-12 (IL-12), which prevented Th2 cell development by promoting T-bet upregulation in responding T cells. Conversely, infants failed to induce TNF-α after HDM + LPS sensitization. Therefore, CD11b+ mDCs failed to upregulate T-bet and did not secrete IL-12 and Th2 cell responses normally developed in infant mice. Thus, the availability of TNF-α dictates the ability of CD11b+ mDCs to suppress allergic Th2-cell responses upon dose-dependent endotoxin sensitization and is a key mediator governing susceptibility to allergic airway inflammation in infant mice.

Dynamic regulation of T follicular regulatory cell responses by interleukin 2 during influenza infection.

Interleukin 2 (IL-2) promotes Foxp3+ regulatory T (Treg) cell responses, but inhibits T follicular helper (TFH) cell development. However, it is not clear how IL-2 affects T follicular regulatory (TFR) cells, a cell type with properties of both Treg and TFH cells. Using an influenza infection model, we found that high IL-2 concentrations at the peak of the infection prevented TFR cell development by a Blimp-1-dependent mechanism. However, once the immune response resolved, some Treg cells downregulated CD25, upregulated Bcl-6 and differentiated into TFR cells, which then migrated into the B cell follicles to prevent the expansion of self-reactive B cell clones. Thus, unlike its effects on conventional Treg cells, IL-2 inhibits TFR cell responses.

CCCTC-Binding Factor Translates Interleukin 2- and α-Ketoglutarate-Sensitive Metabolic Changes in T Cells into Context-Dependent Gene Programs.

Despite considerable research connecting cellular metabolism with differentiation decisions, the underlying mechanisms that translate metabolite-sensitive activities into unique gene programs are still unclear. We found that aspects of the interleukin-2 (IL-2)-sensitive effector gene program in CD4+ and CD8+ T cells in type 1 conditions (Th1) were regulated by glutamine and alpha-ketoglutarate (αKG)-induced events, in part through changes in DNA and histone methylation states. We further identified a mechanism by which IL-2- and αKG-sensitive metabolic changes regulated the association of CCCTC-binding factor (CTCF) with select genomic sites. αKG-sensitive CTCF sites were often associated with loci containing IL-2- and αKG-sensitive genome organization patterns and gene expression in T cells. IL-2- and αKG-sensitive CTCF sites in T cells were also associated with genes from developmental pathways that had αKG-sensitive expression in embryonic stem cells. The data collectively support a mechanism wherein CTCF serves to translate αKG-sensitive metabolic changes into context-dependent differentiation gene programs.

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.

The Integrin LFA-1 Controls T Follicular Helper Cell Generation and Maintenance.

T follicular helper (Tfh) cells are a CD4+ T cell subset critical for long-lived humoral immunity. We hypothesized that integrins play a decisive role in Tfh cell biology. Here we show that Tfh cells expressed a highly active form of leukocyte function-associated antigen-1 (LFA-1) that was required for their survival within the germinal center niche. In addition, LFA-1 promoted expression of Bcl-6, a transcriptional repressor critical for Tfh cell differentiation, and inhibition of LFA-1 abolished Tfh cell generation and prevented protective humoral immunity to intestinal helminth infection. Furthermore, we demonstrated that expression of Talin-1, an adaptor protein that regulates LFA-1 affinity, dictated Tfh versus Th2 effector cell differentiation. Collectively, our results define unique functions for LFA-1 in the Tfh cell effector program and suggest that integrin activity is important in lineage decision-making events in the adaptive immune system.

T Follicular Helper Cell Plasticity Shapes Pathogenic T Helper 2 Cell-Mediated Immunity to Inhaled House Dust Mite.

Exposure to environmental antigens, such as house dust mite (HDM), often leads to T helper 2 (Th2) cell-driven allergic responses. However, the mechanisms underlying the development of these responses are incompletely understood. We found that the initial exposure to HDM did not lead to Th2 cell development but instead promoted the formation of interleukin-4 (IL-4)-committed T follicular helper (Tfh) cells. Following challenge exposure to HDM, Tfh cells differentiated into IL-4 and IL-13 double-producing Th2 cells that accumulated in the lung and recruited eosinophils. B cells were required to expand IL-4-committed Tfh cells during the sensitization phase, but did not directly contribute to disease. Impairment of Tfh cell responses during the sensitization phase or Tfh cell depletion prevented Th2 cell-mediated responses following challenge. Thus, our data demonstrate that Tfh cells are precursors of HDM-specific Th2 cells and reveal an unexpected role of B cells and Tfh cells in the pathogenesis of allergic asthma.

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.