Karine Serre

Extrafollicular antibody responses

Summary:  In adaptive antibody responses, B cells are induced to grow either in follicles where they form germinal centers or in extrafollicular foci as plasmablasts. Extrafollicular growth typically occurs in the medullary cords of lymph nodes and in foci in the red pulp of the spleen. It is not a feature of secondary lymphoid tissue associated with the internal epithelia of the body. All types of naïve and memory B cells can be recruited into extrafollicular responses. These responses are associated with immunoglobulin class switching but, at the most, only low‐level hypermutation.

Dendritic Cells and Monocyte/Macrophages That Create the IL-6/APRIL-Rich Lymph Node Microenvironments Where Plasmablasts Mature

IL-6 and APRIL influence the growth, differentiation, and survival of normal and neoplastic Ab-forming cells (AFC). In this study, we identify two subsets of myeloid cells that associate with the AFC and are the main producers of these factors during a T-dependent Ab response to alum-precipitated protein in mouse lymph nodes. First CD11c+CD8α− dendritic cells located in the perivascular area of the T zone provide about half of the IL-6 mRNA produced in the node together with significant amounts of APRIL mRNA. The number of these cells increases during the response, at least in part due to local proliferation. The second subset comprises Gr1+CD11b+F4/80+ monocyte/macrophages. These colonize the medullary cords during the response and are the other main IL-6 mRNA producers and the greatest source of APRIL mRNA. This medullary cord monocyte/macrophage subset results in local increase of APRIL mRNA that mirrors the polarity of CXCL12 expression in the node. The distribution of these myeloid cell subsets correlates with a gradient of AFC maturation assessed by progressive loss of Ki67 as AFC pass from the B cell follicle along the perivascular areas to the medullary cords.

Salmonella Induces a Switched Antibody Response without Germinal Centers That Impedes the Extracellular Spread of Infection

T-dependent Ab responses are characterized by parallel extrafollicular plasmablast growth and germinal center (GC) formation. This study identifies that, in mice, the Ab response against Salmonella is novel in its kinetics and its regulation. It demonstrates that viable, attenuated Salmonella induce a massive early T-dependent extrafollicular response, whereas GC formation is delayed until 1 mo after infection. The extrafollicular Ab response with switching to IgG2c, the IgG2a equivalent in C57BL/6 mice, is well established by day 3 and persists through 5 wk. Switching is strongly T dependent, and the outer membrane proteins are shown to be major targets of the early switched IgG2c response, whereas flagellin and LPS are not. GC responses are associated with affinity maturation of IgG2c, and their induction is associated with bacterial burden because GC could be induced earlier by treating with antibiotics. Clearance of these bacteria is not a consequence of high-affinity Ab production, for clearance occurs equally in CD154-deficient mice, which do not develop GC, and wild-type mice. Nevertheless, transferred low- and high-affinity IgG2c and less efficiently IgM were shown to impede Salmonella colonization of splenic macrophages. Furthermore, Ab induced during the infection markedly reduces bacteremia. Thus, although Ab does not prevent the progress of established splenic infection, it can prevent primary infection and impedes secondary hemogenous spread of the disease. These results may explain why attenuated Salmonella-induced B cell responses are protective in secondary, but not primary infections.

Virosome-mediated delivery of protein antigens to dendritic cells.

Virosomes are reconstituted viral membranes in which protein can be encapsulated. Fusion-active virosomes, fusion-inactive virosomes and liposomes were used to study the conditions needed for delivery of encapsulated protein antigen ovalbumin (OVA) to dendritic cells (DCs) for MHC class I and II presentation. Fusion-active virosomes, but not fusion-inactive virosomes, were able to deliver OVA to DCs for MHC class I presentation at picomolar OVA concentrations. Fusion activity of virosomes was not required for MHC class II presentation of antigen. Therefore, virosomes are an efficient system for delivery of protein antigens for stimulation of both helper and CTL responses.

Responses to the soluble flagellar protein FliC are Th2, while those to FliC on Salmonella are Th1

Features of the Th1 or Th2 phenotype start to develop during CD4 T cell priming. This study of the response to the bacterial flagellar protein FliC shows that either Th1 or Th2 responses can be induced in mice depending upon how FliC is presented. This is shown by assessing the cytokine mRNA and class of FliC‐specific plasma cells induced in situ. Soluble recombinant (r)FliC and polymerized FliC are strongly Th2 polarizing, inducing IL‐4, NIP45 and c‐Maf mRNA as well as ϵ and γ1 switch transcripts and switching to IgG1. CD28‐requirement for this switching shows its T cell dependence. rFliC was unable to induce markers of Th1 activity including IL‐12, T‐bet and IFN‐γ. Conversely, when FliC is presented in its native context surface‐bound on live, flagellated Salmonella, switching is predominantly to IgG2a (IgG2c in C57BL/6 mice), reflecting Th1 activity. The development of divergent FliC‐specific polarization to either Th1 or Th2 indicates that the context in which this antigen is encountered rather than its intrinsic immunostimulatory properties determines the direction of Th polarization.

Ontogeny of Stromal Organizer Cells during Lymph Node Development

The development of secondary lymphoid organs, such as lymph nodes (LNs), in the embryo results from the reciprocal action between lymphoid tissue inducer (LTi) cells and stromal cells. However, the initial events inducing LN anlagen formation before the LTi stromal cells cross-talk interactions take place are not fully elucidated. In this study, we show that the inguinal LN anlagen in mouse embryos developed from mesenchymal cells surrounding the lymph sacs, spherical structures of endothelial cells that bud from veins. Using inguinal and mesenteric LNs (mLNs), we provide evidence supporting a two-step maturation model for stromal cells: first, ICAM-1−VCAM-1− mesenchymal precursor cells become ICAM-1intVCAM-1int cells, in a process independent of LTi cells and lymphotoxin β receptor (LTβR) signaling. The second step involves the maturation of ICAM-1intVCAM-1int cells to ICAM-1highVCAM-1high mucosal addressin cell adhesion molecule-1+ organizer cells and depends on both LTi cells and LTβR. Addition of αLTβR agonist to LN organ cultures was sufficient to induce ICAM-1intVCAM-1int cells to mature. In LtβR−/− embryos, both inguinal and mLN stromal cells showed a block at the ICAM-1intVCAM-1int stage, and, contrary to inguinal LNs, mLNs persist longer and contained LTi cells, which correlated with the sustained gene expression of Il-7, Cxcl13, and, to a lesser degree, Ccl21. Taken together, these results highlight the importance of the signals and cellular interactions that induce the maturation of stromal cells and ultimately lead to the formation of lymphoid tissues.

Pinpointing IL‐4‐independent acquisition and IL‐4‐influenced maintenance of Th2 activity by CD4 T cells

Naive CD4 T cells develop Th2 activity early in primary responses to alum‐precipitated proteins by producing IL‐4 mRNA and inducing B cells to produce γ1 and ϵ switch transcripts. BothIL‐4‐dependent and IL‐4‐independent pathways for IL‐4 induction are recognized, but their relative contribution to the different phases of primary Th2 responses in vivo is uncertain. We show the primary induction of IL‐4 synthesis in lymph nodes responding to alum‐precipitated protein is overwhelmingly in antigen‐specific CD4 T cells and is unimpaired in IL‐4Rα–/– mice, which can produce but do not respond to IL‐4 and IL‐13. Ig class‐switching in extra‐follicular responses, reflecting Th2 activity, is also unimpaired in these mice. By contrast, 7 days after immunization — when T cells are selecting B cells in germinal centers and T cell priming has occurred — non‐responsiveness to IL‐4 is associated with smaller germinal centers, increased levels of T‐bet andγ2a switch transcripts and reduced γ1 and ϵ transcripts. These data indicate that Th2 characteristics acquired during T cell priming and the initial CD4 T cell interaction with B cells are largely IL‐4‐independent, whereas IL‐4 production induced during priming has a significant role in maintaining the Th2 phenotype as T cells select B cells in germinal centers.

Soluble flagellin, FliC, induces an Ag-specific Th2 response, yet promotes T-bet-regulated Th1 clearance of Salmonella typhimurium infection

Clearance of disseminated Salmonella infection requires bacterial‐specific Th1 cells and IFN‐γ production, and Th1‐promoting vaccines are likely to help control these infections. Consequently, vaccine design has focused on developing Th1‐polarizing adjuvants or Ag that naturally induce Th1 responses. In this study, we show that, in mice, immunization with soluble, recombinant FliC protein flagellin (sFliC) induces Th2 responses as evidenced by Ag‐specific GATA‐3, IL‐4 mRNA, and protein induction in CD62Llo CD4+ T cells without associated IFN‐γ production. Despite these Th2 features, sFliC immunization can enhance the development of protective Th1 immunity during subsequent Salmonella infection in an Ab‐independent, T‐cell‐dependent manner. Salmonella infection in sFliC‐immunized mice resulted in augmented Th1 responses, with greater bacterial clearance and increased numbers of IFN‐γ‐producing CD4+ T cells, despite the early induction of Th2 features to sFliC. The augmented Th1 immunity after sFliC immunization was regulated by T‐bet although T‐bet is dispensable for primary responses to sFliC. These findings show that there can be flexibility in T‐cell responses to some subunit vaccines. These vaccines may induce Th2‐type immunity during primary immunization yet promote Th1‐dependent responses during later infection. This suggests that designing Th1‐inducing subunit vaccines may not always be necessary since this can occur naturally during subsequent infection.

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.

IFN-γ 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.