C-type lectin receptor agonists elicit functional IL21-expressing Tfh cells and induce primary B cell responses in neonates.

Introduction: C-type lectin receptor (CLR) agonists emerged as superior inducers of primary B cell responses in early life compared with Toll-like receptor (TLR) agonists, while both types of adjuvants are potent in adults. Methods: Here, we explored the mechanisms accounting for the differences in neonatal adjuvanticity between a CLR-based (CAF®01) and a TLR4-based (GLA-SE) adjuvant administered with influenza hemagglutinin (HA) in neonatal mice, by using transcriptomics and systems biology analyses. Results: On day 7 after immunization, HA/CAF01 increased IL6 and IL21 levels in the draining lymph nodes, while HA/GLA-SE increased IL10. CAF01 induced mixed Th1/Th17 neonatal responses while T cell responses induced by GLA-SE had a more pronounced Th2-profile. Only CAF01 induced T follicular helper (Tfh) cells expressing high levels of IL21 similar to levels induced in adult mice, which is essential for germinal center (GC) formation. Accordingly, only CAF01- induced neonatal Tfh cells activated adoptively transferred hen egg lysozyme (HEL)-specific B cells to form HEL+ GC B cells in neonatal mice upon vaccination with HEL-OVA. Discussion: Collectively, the data show that CLR-based adjuvants are promising neonatal and infant adjuvants due to their ability to harness Tfh responses in early life.

CD39 and CD326 Are Bona Fide Markers of Murine and Human Plasma Cells and Identify a Bone Marrow Specific Plasma Cell Subpopulation in Lupus.

Antibody-secreting cells (ASCs) contribute to immunity through production of antibodies and cytokines. Identification of specific markers of ASC would allow selective targeting of these cells in several disease contexts. Here, we performed an unbiased, large-scale protein screening, and identified twelve new molecules that are specifically expressed by murine ASCs. Expression of these markers, particularly CD39, CD81, CD130, and CD326, is stable and offers an improved resolution for ASC identification. We accessed their expression in germ-free conditions and in T cell deficient mice, showing that at least in part their expression is controlled by microbial- and T cell-derived signals. Further analysis of lupus mice revealed the presence of a subpopulation of LAG-3- plasma cells, co-expressing high amounts of CD39 and CD326 in the bone marrow. This population was IgM+ and correlated with IgM anti-dsDNA autoantibodies in sera. Importantly, we found that CD39, CD81, CD130, and CD326 are also expressed by human peripheral blood and bone marrow ASCs. Our data provide innovative insights into ASC biology and function in mice and human, and identify an intriguing BM specific CD39++CD326++ ASC subpopulation in autoimmunity.

Stromal Cell-Contact Dependent PI3K and APRIL Induced NF-κB Signaling Prevent Mitochondrial- and ER Stress Induced Death of Memory Plasma Cells.

The persistence of long-lived memory plasma cells in the bone marrow depends on survival factors available in the bone marrow, which are provided in niches organized by stromal cells. Using an ex vivo system in which we supply the known survival signals, direct cell contact to stromal cells, and the soluble cytokine a proliferation-inducing ligand (APRIL), we have elucidated the critical signaling pathways required for the survival of long-lived plasma cells. Integrin-mediated contact of bone marrow plasma cells with stromal cells activates the phosphatidylinositol 3-kinase (PI3K) signaling pathway, leading to critical inactivation of Forkhead-Box-Protein O1/3 (FoxO1/3) and preventing the activation of mitochondrial stress-associated effector caspases 3 and 7. Accordingly, inhibition of PI3K signaling in vivo ablates bone marrow plasma cells. APRIL signaling, by the nuclear factor κB (NF-κB) pathway, blocks activation of the endoplasmic-reticulum-stress-associated initiator caspase 12. Thus, stromal-cell-contact-induced PI3K and APRIL-induced NF-κB signaling provide the necessary and complementary signals to maintain bone marrow memory plasma cells.

Identification of a super-functional Tfh-like subpopulation in murine lupus by pattern perception.

Dysregulated cytokine expression by T cells plays a pivotal role in the pathogenesis of autoimmune diseases. However, the identification of the corresponding pathogenic subpopulations is a challenge, since a distinction between physiological variation and a new quality in the expression of protein markers requires combinatorial evaluation. Here, we were able to identify a super-functional follicular helper T cell (Tfh)-like subpopulation in lupus-prone NZBxW mice with our binning approach "pattern recognition of immune cells (PRI)". PRI uncovered a subpopulation of IL-21+ IFN-γhigh PD-1low CD40Lhigh CXCR5- Bcl-6- T cells specifically expanded in diseased mice. In addition, these cells express high levels of TNF-α and IL-2, and provide B cell help for IgG production in an IL-21 and CD40L dependent manner. This super-functional T cell subset might be a superior driver of autoimmune processes due to a polyfunctional and high cytokine expression combined with Tfh-like properties.

Overcoming the Neonatal Limitations of Inducing Germinal Centers through Liposome-Based Adjuvants Including C-Type Lectin Agonists Trehalose Dibehenate or Curdlan.

Neonates and infants are more vulnerable to infections and show reduced responses to vaccination. Consequently, repeated immunizations are required to induce protection and early life vaccines against major pathogens such as influenza are yet unavailable. Formulating antigens with potent adjuvants, including immunostimulators and delivery systems, is a demonstrated approach to enhance vaccine efficacy. Yet, adjuvants effective in adults may not meet the specific requirements for activating the early life immune system. Here, we assessed the neonatal adjuvanticity of three novel adjuvants including TLR4 (glucopyranosyl lipid adjuvant-squalene emulsion), TLR9 (IC31®), and Mincle (CAF01) agonists, which all induce germinal centers (GCs) and potent antibody responses to influenza hemagglutinin (HA) in adult mice. In neonates, a single dose of HA formulated into each adjuvant induced T follicular helper (TFH) cells. However, only HA/CAF01 elicited significantly higher and sustained antibody responses, engaging neonatal B cells to differentiate into GCs already after a single dose. Although antibody titers remained lower than in adults, HA-specific responses induced by a single neonatal dose of HA/CAF01 were sufficient to confer protection against influenza viral challenge. Postulating that the neonatal adjuvanticity of CAF01 may result from the functionality of the C-type lectin receptor (CLR) Mincle in early life we asked whether other C-type lectin agonists would show a similar neonatal adjuvanticity. Replacing the Mincle agonist trehalose 6,6'-dibehenate by Curdlan, which binds to Dectin-1, enhanced antibody responses through the induction of similar levels of TFH, GCs and bone marrow high-affinity plasma cells. Thus, specific requirements of early life B cells may already be met after a single vaccine dose using CLR-activating agonists, identified here as promising B cell immunostimulators for early life vaccines when included into cationic liposomes.

Exploring the Role of Microbiota in the Limiting of B1 and MZ B-Cell Numbers by Naturally Secreted Immunoglobulins.

Immunoglobulins (Igs)-or antibodies (Ab)-are important to combat foreign pathogens but also to the immune system homeostasis. We developed the AID-/-µS-/- mouse model devoid of total soluble Igs and suitable to monitor the role of Igs on immune homeostasis. We used this experimental system to uncover a negative feedback control of marginal zone (MZ) and B1 B cells numbers by naturally secreted Igs. We raised AID-/-µS-/- mice in germ-free conditions demonstrating that this effect of natural secreted Igs is independent of the microbiota. Herein, we provide a comprehensive description of the protocols to establish and use the AID-/-µS-/- mice to study the role of total secreted Igs or of different Ig classes. This study involves Igs injections to AID-/-µS-/- mice or establishment of AID-/-µS-/- mixed bone marrow chimeras that provide a powerful system to study AID-/-µS-/- B cells in the presence of stable concentrations of different Ig classes. While we describe flow cytometric and histological methods to analyze MZ and B1 B cell subsets, AID-/-µS-/- mice can be used to study the effects of natural Igs on other B cell subsets or immune cells.

Sialic acid removal from dendritic cells improves antigen cross-presentation and boosts anti-tumor immune responses.

Dendritic cells (DCs) hold promise for anti-cancer immunotherapy. However, clinically, their efficiency is limited and novel strategies to improve DC-mediated anti-tumor responses are needed. Human DCs display high content of sialic acids, which inhibits their maturation and co-stimulation capacity. Here, we aimed to understand whether exogenous desialylation of DCs improves their anti-tumor immunity. Compared to fully sialylated DCs, desialylated human DCs loaded with tumor-antigens showed enhanced ability to induce autologous T cells to proliferate, to secrete Th1 cytokines, and to specifically induce tumor cell apoptosis. Desialylated DCs showed an increased expression of MHC-I and -II, co-stimulatory molecules and an augmented secretion of IL-12. Desialylated HLA-A*02:01 DCs pulsed with gp100 peptides displayed enhanced peptide presentation through MHC-I, resulting in higher activation ofgp100280-288 specific CD8+ cytotoxic T cells. Desialylated murine DCs also exhibited increased MHC and co-stimulatory molecules and higher antigen cross-presentation via MHC-I. These DCs showed higher ability to activate antigen-specific CD4+ and CD8+ T cells, and to specifically induce tumor cell apoptosis. Collectively, our data demonstrates that desialylation improves DCs' ability to elicit T cell-mediated anti-tumor activity, due to increased MHC-I expression and higher antigen presentation via MHC-I. Sialidase treatment of DCs may represent a technology to improve the efficacy of antigen loaded-DC-based vaccines for anti-cancer immunotherapy.

Vaccination in early life: standing up to the challenges.

The challenge for any vaccine design is to elicit protective humoral and/or cytotoxic immunity against life threatening pathogens while remaining innocuous. Neonatal vaccinology faces additional challenges linked to intrinsic peculiarities of the innate and adaptive neonatal immune system. These include anti-inflammatory rather than pro-inflammatory responses to innate signals, preferential Th2 differentiation limiting the induction of Th1 and cytotoxic responses, trends to immunoregulatory responses and weak plasma cell and germinal centre B cell responses. Recent progresses in our understanding of the molecular bases of these physiological peculiarities and of the mode of action of novel adjuvants open new opportunities to design vaccine formulations and immunization strategies better adapted to the early life period.

Naturally secreted immunoglobulins limit B1 and MZ B-cell numbers through a microbiota-independent mechanism.

B-cell numbers and immunoglobulin (Ig) titers can increase several logs during immune responses. In contrast to this plasticity and despite constant renewal, B-cell numbers are stable in the absence of immunization. We assessed the role of serum Igs in maintaining specific B-cell subset homeostasis at steady state. Using mice genetically deficient in secreted IgM only (secretory µ chain-deficient), in switched Igs and hypermutated IgM (activation-induced cytidine deaminase-deficient), or fully agammaglobulemic (AID(-/-)µS(-/-)), we dissected the contribution of different Ig classes to 4 phenotypes associated with loss of serum Igs: 1) increased splenic B-cell numbers, mostly of the B1 and marginal zone (MZ) B-cell subtypes; 2) enlarged germinal centers (GCs) in spleen and mesenteric lymph nodes; 3) enrichment in IRF4(+)CD138(-) plasmablast-like cells; and 4) overexpression of IgM in several cell subsets. Complementation experiments based on either mixed bone marrow reconstitution of chimeras or Ig infusion, and analysis of mice raised in germ-free conditions reveal a negative feedback mechanism in which MZ and B1 cell numbers are under the control of naturally secreted Igs as the result of an intrinsic property of the immune system, whereas GC development is under indirect control of secreted Igs that limit bacterial species triggering GC reactions.

CD8 T cells induce T-bet-dependent migration toward CXCR3 ligands by differentiated B cells produced during responses to alum-protein vaccines.

Antibody-forming cells (AFCs) expressing the chemokine receptor CXCR3 are recruited to sites of inflammation where they help clear pathogens but may participate in autoimmune diseases. Here we identify a mechanism that induces CXCR3 expression by AFC and germinal center (GC) B cells. This happens when CD8 T cells are recruited into CD4 T cell-dependent B-cell responses. Ovalbumin-specific CD4 T cells (OTII) were transferred alone or with ovalbumin-specific CD8 T cells (OTI) and the response to subcutaneous alum-precipitated ovalbumin was followed in the draining lymph nodes. OTII cells alone induce T helper 2-associated class switching to IgG1, but few AFC or GC B cells express CXCR3. By contrast, OTI-derived IFN-γ induces most responding GC B cells and AFCs to express high levels of CXCR3, and diverse switching to IgG2a, IgG2b, with some IgG1. Up-regulation of CXCR3 by GC B cells and AFCs and their migration toward its ligand CXCL10 are shown to depend on B cells' intrinsic T-bet, a transcription factor downstream of the IFN-γR signaling. This model clarifies how precursors of long-lived AFCs and memory B cells acquire CXCR3 that causes their migration to inflammatory foci.

A theory of germinal center B cell selection, division, and exit.

High-affinity antibodies are generated in germinal centers in a process involving mutation and selection of B cells. Information processing in germinal center reactions has been investigated in a number of recent experiments. These have revealed cell migration patterns, asymmetric cell divisions, and cell-cell interaction characteristics, used here to develop a theory of germinal center B cell selection, division, and exit (the LEDA model). According to this model, B cells selected by T follicular helper cells on the basis of successful antigen processing always return to the dark zone for asymmetric division, and acquired antigen is inherited by one daughter cell only. Antigen-retaining B cells differentiate to plasma cells and leave the germinal center through the dark zone. This theory has implications for the functioning of germinal centers because compared to previous models, high-affinity antibodies appear one day earlier and the amount of derived plasma cells is considerably larger.

Helios is associated with CD4 T cells differentiating to T helper 2 and follicular helper T cells in vivo independently of Foxp3 expression.

BACKGROUND: Although in vitro IL-4 directs CD4 T cells to produce T helper 2 (Th2)-cytokines, these cytokines can be induced in vivo in the absence of IL-4-signalling. Thus, mechanism(s), different from the in vitro pathway for Th2-induction, contribute to in vivo Th2-differentiation. The pathway for in vivo IL-4-independent Th2-differentiation has yet to be characterized. FINDINGS: Helios (ikzf2), a member of the Ikaros transcription regulator family, is expressed in thymocytes and some antigen-matured T cells as well as in regulatory T cells. It has been proposed that Helios is a specific marker for thymus-derived regulatory T cells. Here, we show that mouse ovalbumin-specific CD4 (OTII) cells responding to alum-precipitated ovalbumin (alumOVA) upregulate Th2 features - GATA-3 and IL-4 - as well as Helios mRNA and protein. Helios is also upregulated in follicular helper T (TFh) cells in this response. By contrast, OTII cells responding to the Th1 antigen - live attenuated ovalbumin-expressing Salmonella - upregulate Th1 features - T-bet and IFN-γ - but not Helios. In addition, CD4 T cells induced to produce Th2 cytokines in vitro do not express Helios. The kinetics of Helios mRNA and protein induction mirrors that of GATA-3. The induction of IL-4, IL-13 and CXCR5 by alumOVA requires NF-κB1 and this is also needed for Helios upregulation. Importantly, Helios is induced in Th2 and TFh cells without parallel upregulation of Foxp3. These findings suggested a key role for Helios in Th2 and TFh development in response to alum-protein vaccines. We tested this possibility using Helios-deficient OTII cells and found this deficiency had no discernable impact on Th2 and TFh differentiation in response to alumOVA. CONCLUSIONS: Helios is selectively upregulated in CD4 T cells during Th2 and TFh responses to alum-protein vaccines in vivo, but the functional significance of this upregulation remains uncertain.

T-zone localized monocyte-derived dendritic cells promote Th1 priming to Salmonella.

Control of intracellular Salmonella infection requires Th1 priming and IFN-γ production. Here, we show that efficient Th1 priming after Salmonella infection requires CD11c(+) CD11b(hi) F4/80(+) monocyte-derived dendritic cells (moDCs). In non-infected spleens, moDCs are absent from T-cell zones (T zones) of secondary lymphoid tissues, but by 24 h post-infection moDCs are readily discernible in these sites. The accumulation of moDCs is more dependent upon bacterial viability than bacterial virulence. Kinetic studies showed that moDCs were necessary to prime but not sustain Th1 responses, while ex vivo studies showed that antigen-experienced moDCs were sufficient to induce T-cell proliferation and IFN-γ production via a TNF-α-dependent mechanism. Importantly, moDCs and cDCs when co-cultured induced superior Th1 differentiation than either subset alone, and this activity was independent of TNF-α. Thus, optimal Th1 development to Salmonella requires the rapid accumulation of moDCs within T zones and their collaboration with cDCs.

Selective effects of NF-κB1 deficiency in CD4⁺ T cells on Th2 and TFh induction by alum-precipitated protein vaccines.

NF-κB1-dependent signaling directs the development of CD4(+) Th2 cells during allergic airway inflammation and protective responses to helminth infection. Here, we show that IL-4 and IL-13 production is NF-κB1-dependent in mouse OVA-specific CD4(+) (OTII) T cells responding to alum-precipitated OVA (alumOVA) immunization. More surprisingly, we found that NF-κB1 deficiency in OTII cells also selectively impairs their CXCR5 induction by alumOVA without affecting upregulation of BCL6, IL-21, OX40 and CXCR4 mRNA and PD-1 protein. This results in functional impairment of follicular helper T cells. Thus, fewer germinal center B cells develop in LN responses to alumOVA in T-cell-deficient mice reconstituted with NF-κB1(-/-) OTII cells as opposed to NF-κB1(+/+) OTII cells, while plasma cell numbers are comparable. Unlike CXCR5 induction in CD4(+) T cells, NF-κB1-deficient recirculating follicular B cells are shown to express normal levels of CXCR5. The selective effects of NF-κB1-deficiency on Th2 and follicular helper T cell induction do not appear to be due to altered expression of the Th2-associated transcription factors - GATA-3, c-Maf and Ikaros. Altogether, these results suggest that NF-κB1 regulates the expression of CXCR5 on CD4(+) T cells primed in vivo, and thus selectively controls the T-cell-dependent germinal center component of B-cell response to alumOVA.

Trypanosoma cruzi infection induces a massive extrafollicular and follicular splenic B-cell response which is a high source of non-parasite-specific antibodies.

Acute infection with Trypanosoma cruzi, the aetiological agent of Chagas' disease, results in parasitaemia and polyclonal lymphocyte activation. It has been reported that polyclonal B-cell activation is associated with hypergammaglobulinaemia and delayed parasite-specific antibody response. In the present study we analysed the development of a B-cell response within the different microenvironments of the spleen during acute T. cruzi infection. We observed massive germinal centre (GC) and extrafollicular (EF) responses at the peak of infection. However, the EF foci were evident since day 3 post-infection (p.i.), and, early in the infection, they mainly provided IgM. The EF foci response reached its peak at 11 days p.i. and extended from the red pulp into the periarteriolar lymphatic sheath. The GCs were detected from day 8 p.i. At the peak of parasitaemia, CD138(+) B220(+) plasma cells in EF foci, red pulp and T-cell zone expressed IgM and all the IgG isotypes. Instead of the substantial B-cell response, most of the antibodies produced by splenic cells did not target the parasite, and parasite-specific IgG isotypes could be detected in sera only after 18 days p.i. We also observed that the bone marrow of infected mice presented a strong reduction in CD138(+) B220(+) cells compared with that of normal mice. Hence, in acute infection with T. cruzi, the spleen appears to be the most important lymphoid organ that lodges plasma cells and the main producer of antibodies. The development of a B-cell response during T. cruzi infection shows features that are particular to T. cruzi and other protozoan infection but different to other infections or immunization with model antigens.

IFN-{gamma} produced by CD8 T cells induces T-bet-dependent and -independent class switching in B cells in responses to alum-precipitated protein vaccine.

Alum-precipitated protein (alum protein) vaccines elicit long-lasting neutralizing antibody responses that prevent bacterial exotoxins and viruses from entering cells. Typically, these vaccines induce CD4 T cells to become T helper 2 (Th2) cells that induce Ig class switching to IgG1. We now report that CD8 T cells also respond to alum proteins, proliferating extensively and producing IFN-γ, a key Th1 cytokine. These findings led us to question whether adoptive transfer of antigen-specific CD8 T cells alters the characteristic CD4 Th2 response to alum proteins and the switching pattern in responding B cells. To this end, WT mice given transgenic ovalbumin (OVA)-specific CD4 (OTII) or CD8 (OTI) T cells, or both, were immunized with alum-precipitated OVA. Cotransfer of antigen-specific CD8 T cells skewed switching patterns in responding B cells from IgG1 to IgG2a and IgG2b. Blocking with anti-IFN-γ antibody largely inhibited this altered B-cell switching pattern. The transcription factor T-bet is required in B cells for IFN-γ-dependent switching to IgG2a. By contrast, we show that this transcription factor is dispensable in B cells both for IFN-γ-induced switching to IgG2b and for inhibition of switching to IgG1. Thus, T-bet dependence identifies distinct transcriptional pathways in B cells that regulate IFN-γ-induced switching to different IgG isotypes.

Megakaryocytes constitute a functional component of a plasma cell niche in the bone marrow.

Long-lived plasma cells in the bone marrow produce memory antibodies that provide immune protection persisting for decades after infection or vaccination but can also contribute to autoimmune and allergic diseases. However, the composition of the microenvironmental niches that are important for the generation and maintenance of these cells is only poorly understood. Here, we demonstrate that, within the bone marrow, plasma cells interact with the platelet precursors (megakaryocytes), which produce the prominent plasma cell survival factors APRIL (a proliferation-inducing ligand) and IL-6 (interleukin-6). Accordingly, reduced numbers of immature and mature plasma cells are found in the bone marrow of mice deficient for the thrombopoietin receptor (c-mpl) that show impaired megakaryopoiesis. After immunization, accumulation of antigen-specific plasma cells in the bone marrow is disturbed in these mice. Vice versa, injection of thrombopoietin allows the accumulation and persistence of a larger number of plasma cells generated in the course of a specific immune response in wild-type mice. These results demonstrate that megakaryocytes constitute an important component of the niche for long-lived plasma cells in the bone marrow.

IL-4 directs both CD4 and CD8 T cells to produce Th2 cytokines in vitro, but only CD4 T cells produce these cytokines in response to alum-precipitated protein in vivo.

While IL-4 directs CD4 T cells to produce Th2 cytokines (including IL-4, IL-13, IL-5) in vitro it has been shown that production of these cytokines can be induced in vivo in the absence of IL-4/IL-13/STAT-6 signaling. The present report shows that CD8 as well as CD4 T cells activated through their TCR, in vitro upregulate the Th2-features - IL-4, IL-13, IL-5, and GATA-3. However, in vivo while alum-precipitated antigen strongly and selectively induces these Th2-features in CD4 T cells, CD8 T cells mount a markedly different response to this antigen. This CD8 response is associated with strong proliferation and production of IFN-gamma, but no Th2-features are induced. Alum-protein formulations are widely used in human vaccines and typically induce strong antibody responses characterized by the differentiation of IL-4-producing CD4 T cells and immunoglobulin class switching to IgG1. Nevertheless, the mechanism responsible for CD4 Th2 and follicular helper T cell commitment triggered by these alum-protein vaccines is still poorly understood. Analysis of the in vivo response to alum-precipitated protein shows that while subsets of CD4 T cells strongly upregulate Th2 and follicular helper T cell features including the surface markers OX40, CXCR5, PD-1, IL-17RB and the transcription factor c-Maf, CD8 T cells do not. These discrete differences between responding CD4 and CD8 T cells provide further insight into the differences between Th2 polarization of CD4 T cells directed by IL-4 in vitro and the induction of IL-4 production by CD4 T cells in vivo in response to alum-precipitated protein.