Search

Germinal center (GC)-derived memory B cells (MBCs) are critical for humoral immunity as they differentiate into protective antibody-secreting cells during re-infection. GC formation and cellular interactions within the GC have been studied in detail, yet the exact signals that allow for the selection and exit of MBCs are not understood. Here, we showed that IL-4 cytokine signaling in GC B cells directly downregulated the transcription factor BCL6 via negative autoregulation to release cells from the GC program and to promote MBC formation. This selection event required additional survival cues and could therefore result in either GC exit or death. We demonstrate that both increasing IL-4 bioavailability or limiting IL-4 signaling disrupted MBC selection stringency. In this way, IL-4 control of BCL6 expression serves as a tunable switch within the GC to tightly regulate MBC selection and affinity maturation., Competing Interests: Declaration of interests M.P. is a member of the Vaxart scientific advisory board., (Copyright © 2024 Elsevier Inc. All rights reserved.)

CD4+ lung-resident memory T cells (TRM) generated in response to influenza infection confer effective protection against subsequent viral exposures. Whether these cells can be altered by environmental antigens and cytokines released during heterologous, antigen-independent immune responses is currently unclear. We therefore investigated how influenza-specific CD4+ Th1 TRM in the lung are impacted by a subsequent Th2-inducing respiratory house dust mite (HDM) exposure. Although naïve influenza-specific CD4+ T cells in the lymph nodes do not respond to HDM, influenza-specific CD4+ TRM in the lungs do respond to a subsequent allergen exposure by decreasing expression of the transcription factor T-bet. This functional alteration is associated with decreased IFN-γ production upon restimulation and improved disease outcomes following heterosubtypic influenza challenge. Further investigation revealed that ST2 signaling in CD4+ T cells during allergic challenge is necessary to induce these changes in lung-resident influenza-specific CD4+ TRM. Thus, heterologous antigen exposure or ST2-signaling can drive persistent changes in CD4+ Th1 TRM populations and impact protection upon reinfection., (© 2023 Rüterbusch et al.)

Germinal center (GC)-derived memory B cells (MBCs) are critical for humoral immunity as they differentiate into protective antibody-secreting cells during re-infection. GC formation and cellular interactions within the GC have been studied in detail, yet the exact signals that allow for the selection and exit of MBCs are not understood. Here, we show that IL-4 signaling in GC B cells directly downregulates BCL6 via negative autoregulation to release cells from the GC program and promote MBC formation. This selection event requires additional survival cues and can therefore result in either GC exit or death. We demonstrate that both increasing IL-4 bioavailability or limiting IL-4 signaling disrupt MBC selection stringency. In this way, IL-4 control of BCL6 expression serves as a tunable switch within the GC to tightly regulate MBC selection and affinity maturation.

CD4 + tissue resident cells are an important first line of defense against viral infections in the lungs and are critical for promoting the localization of lung resident CD8 + T cells. However, relatively little is known about the signaling programs required for the development of viral-specific CD4 + tissue resident cells in the lungs. Recently, it was shown that signaling through the high affinity IL-2 receptor is required for the differentiation of lung-resident Th2 memory (Trm) cells in a murine model of airway inflammation. We therefore tested if IL-2 signaling is also required for the development of viral antigen-specific CD4 + Th1 cells in the lung after i.n. infection with lymphocytic choriomeningitis virus. These studies demonstrate that Th1 CD4 + T cells also require IL-2 for lung Trm development. Additionally, they show that B cells potently inhibit early Th1 cell lung residency, but are required for the maintenance of a long-lived population of CD4 + Th1 Trm., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Many current malaria vaccines target the pre-erythrocytic stage of infection in the liver. However, in malaria-endemic regions, increased blood stage exposure is associated with decreased vaccine efficacy, thereby challenging current vaccine efforts. We hypothesized that pre-erythrocytic humoral immunity is directly disrupted by blood stage infection. To investigate this possibility, we used Plasmodium-antigen tetramers to analyze B cells after infection with either late liver stage arresting parasites or wild-type parasites that progress to the blood stage. Our data demonstrate that immunoglobulin G (IgG) antibodies against the pre-erythrocytic antigen, circumsporozoite protein (CSP), are generated only in response to the attenuated, but not the wild-type, infection. Further analyses revealed that blood stage malaria inhibits CSP-specific germinal center B cell differentiation and modulates chemokine expression. This results in aberrant memory formation and the loss of a rapid secondary B cell response. These data highlight how immunization with attenuated parasites may drive optimal immunity to malaria., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Exposure to inhaled allergens generates T helper 2 (Th2) CD4(+) T cells that contribute to episodes of inflammation associated with asthma. Little is known about allergen-specific Th2 memory cells and their contribution to airway inflammation. We generated reagents to understand how endogenous CD4(+) T cells specific for a house dust mite (HDM) allergen form and function. After allergen exposure, HDM-specific memory cells persisted as central memory cells in the lymphoid organs and tissue-resident memory cells in the lung. Experimental blockade of lymphocyte migration demonstrated that lung-resident cells were sufficient to induce airway hyper-responsiveness, which depended upon CD4(+) T cells. Investigation into the differentiation of pathogenic Trm cells revealed that interleukin-2 (IL-2) signaling was required for residency and directed a program of tissue homing migrational cues. These studies thus identify IL-2-dependent resident Th2 memory cells as drivers of lung allergic responses., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The identification of novel T cell antigens is central to basic and translational research in autoimmunity, tumor immunology, transplant immunology, and vaccine design for infectious disease. However, current methods for T cell antigen discovery are low throughput, and fail to explore a wide range of potential antigen-receptor interactions. To overcome these limitations, we developed a method in which programmable microarrays are used to cost-effectively synthesize complex libraries of thousands of minigenes that collectively encode the content of hundreds of candidate protein targets. Minigene-derived mRNA are transfected into autologous antigen presenting cells and used to challenge complex populations of purified peripheral blood CD8+ T cells in multiplex, parallel ELISPOT assays. In this proof-of-concept study, we apply synthetic minigene screening to identify two novel pancreatic islet autoantigens targeted in a patient with Type I Diabetes. To our knowledge, this is the first successful screen of a highly complex, synthetic minigene library for identification of a T cell antigen. In principle, responses against the full protein complement of any tissue or pathogen can be assayed by this approach, suggesting that further optimization of synthetic libraries holds promise for high throughput antigen discovery.

T regulatory cells are critical for the prevention of autoimmunity. Specifically, Treg cells can control anti-chromatin antibody production in vivo, and this correlates with decreased ICOS expression on CD4(+) T helper cells. Here we test the significance of high ICOS expression by T effector cells, firstly in terms of the anti-chromatin B cell response, and secondly on the ability of Treg cells to suppress T cell help. We bred CD4(+) T cell receptor transgenic mice with mice that carry the Roquin(san/san) mutation. The Roquin gene functions to limit ICOS mRNA such that CD4 T cells from mutant mice express elevated ICOS. Using an in vivo model, TS1.Roquin(san/san) Th cells were compared with wild-type TS1 Th cells with regard to their ability to help anti-chromatin B cells in the presence or absence of Treg cells. Both TS1 and TS1.Roquin(san/san) Th cells induced anti-chromatin IgM(a) antibodies, but the TS1.Roquin(san/san) Th cells resulted in the recovery of more class-switched and germinal center B cells. Neither source of Th cells were capable of inducing long-lived autoantibodies. Treg cells completely suppressed anti-chromatin IgM(a) antibody production and reduced anti-chromatin B cell recovery induced by TS1 Th cells. Importantly, this suppression was less effective when TS1.Roquin(san/san) Th cells were used. Thus, high ICOS levels on effector T cells results in autoimmunity by augmenting the autoreactive B cell response and by dampening the effect of Treg cell suppression.

T-cell recognition of peptide/MHC complexes is flexible and can lead to differential activation, but how interactions with agonist (full activation) or partial agonist (suboptimal activation) peptides can shape immune responses in vivo is not well characterized. We investigated the effect of stimulation by agonist or partial agonist ligands during initial CD4(+) T-cell priming, and subsequent T-B-cell cognate interactions, on antibody production by anti-chromatin B cells. We found that autoantibody production required TCR recognition of an agonist peptide at the effector stage of B-cell activation. However, interaction with a weak agonist ligand at this effector stage failed to promote efficient autoantibody production, even if the CD4(+) T cells were fully primed by an agonist peptide. These studies suggest that the reactivity of the TCR for a target self-peptide during CD4(+) T-B-cell interaction can be a critical determinant in restraining anti-chromatin autoantibody production.

In Fas/FasL-deficient mice anti-chromatin Ab production is T cell dependent and is not apparent until after 10 weeks of age. Early control of anti-chromatin antibodies may be due to the counterbalancing influence of Treg cells. Here we show that Treg cells block lpr/lpr gld/gld Th cells from providing help to anti-chromatin B cells in an in vivo transfer system. Interestingly, the percentage and absolute numbers of Foxp3+ Treg cells is elevated in BALB/c-lpr/lpr gld/gld mice and increases with age compared to BALB/c mice. The majority of Foxp3 expression is found in the B220- CD4+ T cell population, and Foxp3-expressing cells are localized in the splenic PALS (periarteriolar lymphocyte sheath). Strikingly, although the lack of functional Fas/FasL does not affect the ability of Treg cells to block Th cell proliferation, Treg cells can block the IFN-gamma differentiation of Th cells from BALB/c or young BALB-lpr/lpr gld/gld mice but not of pre-existing Th1 cells from older BALB/c-lpr/lpr gld/gld mice. Thus, we suggest autoantibody production is not caused by the lack of Treg cells but by a defect in activation-induced cell death that leads to the accumulation of T effector cells that are resistant to regulatory T cell activity.

B lymphocyte stimulator (BLyS), also known as B cell-activating factor, is a key positive regulator of B cell homeostasis, and elevated levels of BLyS have been observed in systemic lupus erythematosus (SLE) patients. Given that anti-chromatin auto-antibodies are one of the hallmarks of SLE, we examined the role of BLyS and its receptors in the regulation of anti-chromatin B cells. We demonstrate that exogenous BLyS treatment leads to an increase in B cell numbers, particularly anti-chromatin B cells; yet, their localization in the spleen and auto-antibody production remain unaffected. We also examined transmembrane activator and CAML interactor (TACI), BLyS receptor 3 (BR3) and B cell maturation antigen expression on anti-chromatin B cells before and after receiving T cell help. Interestingly, in the absence of T cell help, TACI expression is greater on immature anti-chromatin B cells compared with immature Tg(-) B cells, whereas BR3 levels are comparable. After receiving T cell help, the anti-chromatin B cells that have differentiated into short-lived plasma cells no longer express BR3 but retain TACI. These data suggest a novel role for TACI in anti-chromatin B cell homeostasis and differentiation.

We have taken an immunoglobulin transgenic approach to study how self-reactive B cells are held in check in healthy mice and what parameters contribute to their activation in autoimmunity. Using this strategy, we have documented that a population of anti-chromatin B cells migrate to the periphery. In a healthy background, these cells have a reduced lifespan, appear developmentally arrested, and localize primarily to the T/B cell interface in the spleen. Importantly, they are capable of differentiating into antibody-forming cells when provided with T cell help. T(H)1 and T(H)2 cells induce IgG2a and IgG1 autoantibodies, respectively. In the context of the autoimmune-prone lpr/lpr or gld/gld mutations, these autoreactive B cells populate the B cell follicle, and this is dependent upon CD4 T cells. However, after 10 weeks of age serum autoantibodies are produced. We hypothesize that control of autoantibody production in young autoimmune-prone mice is regulated by the counterbalancing influence of regulatory T cells. We show that while autoantibody production is blocked in the context of regulatory T cells, early events characterizing a productive T cell-B cell interaction are not disturbed, with the notable exceptions of T(H) ICOS levels and IFN-gamma and IL-10 production.

Autoreactive B cells may become activated in a T-independent manner via synergistic engagement of the BCR and TLRs. Using the VH3H9 Ig H chain transgene to track anti-chromatin B cells, we demonstrate that VH3H9/Vlambda1 anti-chromatin B cells proliferate in response to stimulatory oligodeoxynucleotides containing CpG motifs, suggesting that these autoreactive B cells are responsive to TLR9 signaling. Strikingly, some VH3H9 B cells, but not the well-characterized VH3H9/Vlambda1 B cells, proliferate spontaneously in culture medium. This proliferation is blocked by inhibitory CpG oligodeoxynucleotides, implicating the TLR9 (or possibly TLR7) pathway. Most hybridomas generated from the proliferating cells are polyreactive, and one exhibits binding to nuclear Ags but not to the other Ags tested. Thus, B cells carrying autoreactive and/or polyreactive specificities may be susceptible to T cell-independent activation via dual engagement of the BCR and TLRs.

To investigate the mechanism by which T regulatory (Treg) cells may control the early onset of autoimmunity, we have used an adoptive transfer model to track Treg, Th, and anti-chromatin B cell interactions in vivo. We show that anti-chromatin B cells secrete Abs by day 8 in vivo upon provision of undeviated, Th1- or Th2-type CD4+ T cell help, but this secretion is blocked by the coinjection of CD4+ CD25+ Treg cells. Although Treg cells do not interfere with the initial follicular entry or activation of Th or B cells at day 3, ICOS levels on Th cells are decreased. Furthermore, Treg cells must be administered during the initial phases of the Ab response to exert full suppression of autoantibody production. These studies indicate that CD25+ Treg cells act to inhibit the maturation, rather than the initiation, of autoantibody responses.

Circulating autoantibodies against dsDNA and chromatin are a characteristic of systemic lupus erythematosus in humans and many mouse models of this disease. B cells expressing these autoantibodies are normally regulated in nonautoimmune-prone mice but are induced to secrete Abs following T cell help. Likewise, anti-chromatin autoantibody production is T cell-dependent in Fas/Fas ligand (FasL)-deficient (lpr/lpr or gld/gld) mice. In this study, we demonstrate that Th2 cells promote anti-chromatin B cell survival and autoantibody production in vivo. FasL influences the ability of Th2 cells to help B cells, as Th2-gld/gld cells support higher titers of anti-chromatin Abs than their FasL-sufficient counterparts and promote anti-chromatin B cell participation in germinal centers. Th1 cells induce anti-chromatin B cell germinal centers regardless of FasL status; however, their ability to stimulate anti-chromatin Ab production positively correlates with their level of IFN-gamma production. This distinction is lost if FasL-deficient T cells are used: Th1-gld/gld cells promote significant titers of anti-chromatin Abs regardless of IFN-gamma production levels. Thus, FasL from effector T cells plays an important role in determining the fate of anti-chromatin B cells.

Anti-double-stranded DNA (anti-dsDNA) B cells are regulated in non-autoimmune mice. While some are deleted or undergo receptor editing, a population of anti-dsDNA (VH3H9/V lambda 1) B cells that emigrate into the periphery has also been identified. These cells have an altered phenotype relative to normal B cells in that they have a reduced lifespan, appear developmentally arrested, and localize primarily to the T/B-cell interface in the spleen. This phenotype may be the consequence of immature B cells encountering antigen in the absence of T-cell help. When provided with T-cell help, the anti-dsDNA B cells differentiate into antibody-forming cells. In the context of the autoimmune-prone lpr/lpr or gld/gld mutations, the VH3H9/V lambda 1 anti-dsDNA B cells populate the B-cell follicle and by 12 weeks of age produce serum autoantibodies. The early event of anti-dsDNA B-cell follicular entry, in the absence of autoantibody production, is dependent upon CD4(+) T cells. We hypothesize that control of autoantibody production in young autoimmune-prone mice may be regulated by the counterbalancing effect of T-regulatory (T(reg)) cells. Consistent with this model, we have demonstrated that T(reg) cells are able to prevent autoantibody production induced by T-cell help. Additional studies are aimed at investigating the mechanisms of this suppression as well as probing the impact of distinct forms of T-cell-dependent and -independent activation on anti-dsDNA B cells.

IL-12 initiates Th1 cell development and cell-mediated immunity, but whether IL-12 contributes to the maintenance of a Th1 response is unclear. To address this question, we infected IL-12 p40-/- C57BL/6 mice with Leishmania major, an intracellular protozoan parasite controlled by a cell-mediated immune response, and simultaneously administered IL-12. Whereas untreated p40-/- mice developed an uncontrolled infection, p40-/- mice treated with IL-12 for the first 2 or 4 wk of infection developed a Th1 response and resolved their lesions. However, the induction of this protective Th1 cell response by IL-12 treatment was not associated with long term immunity. We observed that on rechallenge in the absence of IL-12, the mice exhibited a susceptible phenotype. In addition, without rechallenge, lesions in the IL-12-treated p40-/- mice developed several weeks after cessation of IL-12 treatment. In both cases, disease was associated with the loss of a Th1 response and the development of a Th2 response. Our observations are not limited to the C57BL/6 strain, because IL-12 treatment was also unable to provide lasting protection to p40-/- BALB/c mice. Finally, we found that although Th1 cells from healed wild-type C57BL/6 mice adoptively transferred protection to L. major-infected RAG-/- mice, they were unable to protect p40-/- mice. In conclusion, these studies provide the first demonstration that IL-12 is required not only to initiate Th1 cell development but also throughout infection to maintain a Th1 cell response and resistance to L. major.

BALB/c and anti-IL-12-treated C3H mice infected with Leishmania major develop a Th2 cell response. However, in contrast to BALB/c mice, C3H mice treated transiently with an anti-IL-12 monoclonal antibody switch from a Th2 to a Th1 response and resolve their lesions once treatment is terminated. We report here that the critical difference in the Th2 response between BALB/c and C3H mice is in their ability to respond to IL-12. Thus, C3H mice with a Th2 response maintain a CD4+ T cell population that expresses IL-12 receptor beta1 and beta2 mRNA and produces IFN-gamma after exposure to IL-12. These results indicate that Th2 cell populations from different genetic backgrounds differ in their stability, and that this difference can be related to differential regulation of the IL-12 receptor.

These studies were undertaken with the purpose of elucidating the key signals involved in the regulation of the production of soluble interleukin-4 receptors (sIL-4R) in mice during Th1 and Th2 responses to infection with the parasite Leishmania major. Our results showed that the production of sIL-4R was consistently higher in lymph node cell cultures from animals mounting a predominant Th2 response (BALB/c mice), and that sIL-4R production paralleled that of IL-4 in both mouse strains, even in the presence of a dominant Th1 response (C3H/FeJ mice). Consistently, administration of anti-IL-12 antibodies to infected C3H/ FeJ mice induced a switch from a Th1- to a Th2-type response and resulted in enhanced production of sIL-4R. Addition of rIL-12 to splenic cell cultures, however, was found not to have a direct effect on sIL-4R production induced by IL-4 or T cell mitogens. Moreover, the production of sIL-4R appears to be little influenced by Th1-produced cytokines, inasmuch as recombinant interferon-gamma or supernatants derived from antigen-stimulated Th1 clones did not affect the production of sIL-4R by activated splenic cultures. Despite its correlation with Th2 responses, the presence of IL-4 was not an absolute requirement for the up-regulation of the expression of sIL-4R because increased levels could be induced on cells obtained from IL-4-/- mice. These results indicate that, although enhanced sIL-4R production is a feature related to the activation and/or generation of Th2 responses, it is not absolutely dependent on IL-4 or directly inhibited by IL-12 or Th1 cytokines.

IL-12 initiates the development of cell-mediated immunity by promoting the differentiation of naive T cells into the Th1 phenotype, and is essential in the development of a Th1 immune response to the intracellular protozoan parasite, Leishmania major. The present study investigated whether IL-12 is also required for the maintenance and effector function of an established Th1 immune response in L. major-infected mice. While neutralization of IL-12 compromised the ability of a leishmanial antigen-reactive Th1 cell clone to produce IFN-gamma in vitro, lymph node cells taken from 2-week L. major-infected mice were able to secrete IFN-gamma in an IL-12-independent manner. However, when a short-term T cell line was established in vitro from lymph node cells, the production of IFN-gamma again became IL-12 dependent. These results suggest that other factors may compensate for IL-12 in vivo in promoting IFN-gamma production during L. major infection. To directly assess if IL-12 was required in vivo for resistance to L. major, we studied the effect of IL-12 neutralization on both a primary and secondary L. major infection in C3H mice. L. major infection in C3H mice is characterized by the development of a small lesion that heals by 8 weeks, and these animals are resistant to reinfection. As previously reported, administration of anti-IL-12 monoclonal antibody (mAb) during a primary infection led to severe disease. However, mice that had healed from a primary infection with L. major and were treated with anti-IL-12 mAb were as resistant as control animals. These findings suggest that once Th1 cells have developed, their effector function in vivo is independent of IL-12, and that this independence is not due to an intrinsic property of the T cell, but to the microenvironment created by the infection.

Leishmania major-infected C3H mice develop a Th1 response, but studies have shown that treatment of C3H mice with anti-IL-12 or anti-IFN-gamma mAb promotes the development of a Th2 response and susceptibility. However, we discovered that C3H mice treated for 3 wk with either anti-IL-12 or anti-IFN-gamma mAb eventually resolved their lesions and switched from a Th2 to a Th1 response. No significant differences in IL-4, IL-10, or IFN-gamma levels or in the parasite burden could be detected between BALB/c and anti-IL-12-treated C3H mice early after infection, suggesting that the instability of the Th2 response in anti-IL-12-treated C3H mice was unrelated to levels of these cytokines and parasite numbers. However, anti-IL-12-treated C3H mice continued to produce IL-12 in spite of exhibiting a Th2 phenotype. To determine whether the production of IL-12 was associated with the healing observed in these animals, we treated C3H mice with anti-IL-12 continuously for 12 wk. In contrast to C3H mice given anti-IL-12 for 3 wk, C3H mice continuously treated with anti-IL-12 failed to heal. These results suggest that qualitative differences in Th2-type responses may influence their stability and that the presence of IL-4, IL-10, or high parasite numbers is not sufficient to maintain a Th2 response in mice with certain genetic backgrounds.

Experimental infections of mice with the protozoan parasite Leishmania major provide an excellent model for defining the conditions required for generation of CD4+ Th1 and Th2 cells in vivo. Since interleukin-12 (IL-12) has been implicated in the development of Th1 cells, we investigated whether L. major stimulates IL-12 production in vitro or in vivo. Surprisingly, macrophages cultured in vitro failed to produce IL-12 following L. major infection. In contrast, lymph node cells from C3H mice infected for 2 days with L. major produced elevated levels of IL-12. In order to determine if the inability to stimulate IL-12 production was limited to in vitro infections, we infected macrophages in vivo by inoculating L. major into the peritoneal cavity. Peritoneal cells isolated 24 h later exhibited a significant increase in the number of cells producing IL-12. In addition, supernatants harvested from these cells following culture contained elevated levels of IL-12. These data indicate that L. major infection induces increased IL-12 production in mice.