Chantal Tougne

APRIL is critical for plasmablast survival in the bone marrow and poorly expressed by early-life bone marrow stromal cells.

The persistence of serum IgG antibodies elicited in human infants is much shorter than when such responses are elicited later in life. The reasons for this rapid waning of antigen-specific antibodies elicited in infancy are yet unknown. We have recently shown that adoptively transferred tetanus toxoid (TT)-specific plasmablasts (PBs) efficiently reach the bone marrow (BM) of infant mice. However, TT-specific PBs fail to persist in the early-life BM, suggesting that they fail to receive the molecular signals that support their survival/differentiation. Using a proliferation-inducing ligand (APRIL)- and B-cell activating factor (BAFF) B-lymphocyte stimulator (BLyS)-deficient mice, we demonstrate here that APRIL is a critical factor for the establishment of the adult BM reservoir of anti-TT IgG-secreting cells. Through in vitro analyses of PB/plasma cell (PC) survival/differentiation, we show that APRIL induces the expression of Bcl-X(L) by a preferential binding to heparan sulfate proteoglycans at the surface of CD138(+) cells. Last, we identify BM-resident macrophages as the main cells that provide survival signals to PBs and show that this function is slowly acquired in early life, in parallel to a progressive acquisition of APRIL expression. Altogether, this identifies APRIL as a critical signal for PB survival that is poorly expressed in the early-life BM compartment.

Determinants of infant responses to vaccines in presence of maternal antibodies.

Presence of maternally-derived antibodies at time of immunization is known to often interfere with active infant immunization, although with variable degrees of clinical significance. In order to progressively decipher the rules that form the basis for these inhibitory effects on infant vaccine responses, two antigens (measles, tetanus) and various antigen presentation systems were evaluated in murine early life immunization models either in absence or presence of maternal antibodies. Both conventional (proteins, conjugate vaccines) and new (live viral vectors, DNA plasmids) antigen presentation systems were found to be similarly susceptible to the inhibitory influence of maternal antibodies. Factors emerging as crucial determinants of maternal antibody-mediated effects on responses to both live and non-live vaccines include (i) the level of maternal antibodies present at immunization, (ii) the use of distinct vaccines in mothers and pups and (iii) their distinct influence on B cell and T cell vaccine responses.

Unresponsiveness to Lymphoid-Mediated Signals at the Neonatal Follicular Dendritic Cell Precursor Level Contributes to Delayed Germinal Center Induction and Limitations of Neonatal Antibody Responses to T-Dependent Antigens

The factors limiting neonatal and infant IgG Ab responses to T-dependent Ags are only partly known. In this study, we assess how these B cell responses are influenced by the postnatal development of the spleen and lymph node microarchitecture. When BALB/c mice were immunized with alum-adsorbed tetanus toxoid at various stages of their immune development, a major functional maturation step for induction of serum IgG, Ab-secreting cells, and germinal center (GC) responses was identified between the second and the third week of life. This correlated with the development of the follicular dendritic cell (FDC) network, as mature FDC clusters only appeared at 2 wk of age. Adoptive transfer of neonatal splenocytes into adult SCID mice rapidly induced B cell follicles and FDC precursor differentiation into mature FDC, indicating effective recruitment and signaling capacity of neonatal B cells. In contrast, adoptive transfer of adult splenocytes into neonatal SCID mice induced primary B cell follicles without any differentiation of mature FDC and failed to correct limitations of tetanus toxoid-induced GC. Thus, unresponsiveness to lymphoid-mediated signals at the level of neonatal FDC precursors delays FDC maturation and GC induction, thus limiting primary Ab-secreting cell responses to T-dependent Ags in early postnatal life.

Delayed and deficient establishment of the long‐term bone marrow plasma cell pool during early life

Early life antibody responses are characterized by a rapid decline, such that antigen‐specific IgG antibodies decline to baseline levels within months following infant immunization. This generic observation remains unexplained. Here, we have analyzed the induction and organ‐localization of antigen‐specific IgG antibody‐secreting cells (ASC) following immunization of 1‐week‐old or adult BALB/c mice with tetanus toxoid (TT), a T‐dependent antigen. Early life priming induced only slightly lower numbers of TT‐specific IgG ASC in the spleen, and these reached adult levels following repeat immunization. In contrast, early life immunization generated much fewer bone marrow plasma cells than in adults, even after boosting. A similar limitation of the natural development of the bone marrow pool of ASC was observed. Transfer experiments with adult or early life spleen ASC indicated limited homing of TT‐specific adult ASC to the bone marrow of 4‐week‐old mice as compared to adult recipients, whereas homing patterns were similar when early life or adult ASC were transferred into adult recipients. These observations suggest that a limited bone marrow B cell homing capacity and, as a result, relatively deficient bone marrow ASC responses, are critical factors which may explain the limited persistence of IgG antibodies to T‐dependent antigens in early life.

Homing and Adhesion Patterns Determine the Cellular Composition of the Bone Marrow Plasma Cell Niche

According to commonly held concepts, plasma cell (PC) longevity in bone marrow (BM) depends upon their access to survival niches. These are thought to exist in nursery cell types, which support PCs by secreting PC survival factors. To better define PC survival niches and their functioning, we adoptively transferred traceable Blimp-1-GFP PCs into recipient mice lacking a proliferation-inducing ligand (APRIL), IL-6, or macrophage migration inhibitory factor. Transferred BMPCs were preferentially associated with Ly-6Chigh monocytes (normalized colocalization index: 9.84), eosinophils (4.29), and megakaryocytes (2.12). Although APRIL was essential for BMPC survival, PC recruitment into the proximity of nursery cells was unimpaired in APRIL-deficient mice, questioning the concept that the same factors account for attraction/retention of PCs as for their local survival. Rather, the order of colocalization with BMPCs (monocytes > eosinophils > megakaryocytes) reflected these cells’ relative expression of CXCR4, VLA-4, and LFA-1, the homing and adhesion molecules that direct/retain PCs in the BM. This suggests a scenario wherein the cellular composition of the BMPC niche is defined by a common pattern of attraction/retention on CXCL12-abundant reticular docking cells. Thereby, PCs are directed to associate in a functional BM niche with hematopoietic CXCR4+VLA-4+LFA-1+ nursery cells, which provide PC survival factors.

Reduced Ability of Neonatal and Early-Life Bone Marrow Stromal Cells to Support Plasmablast Survival

In human infants (<1 year), circulating IgG Abs elicited in response to most T-dependent Ags rapidly decline and return to baseline within a few months after immunization for yet-unknown reasons. In mice immunized between 1 and 4 wk of age, a limited establishment of the bone marrow (BM) pool of long-lived plasma cells is observed. In this study, we show that tetanus toxoid (TT)-specific plasmablasts generated in the spleen are efficiently attracted in vitro and in vivo toward early-life BM stromal cells, which express adult levels of CXCL12. Similarly, adoptively transferred TT plasmablasts efficiently reach the BM compartment of 2-wk-old and adult mice. In contrast, TT plasmablasts fail to persist in the early-life BM compartment, as indicated by the persistence of a significantly lower number of TT plasmablasts in the early-life compartment than in the adult BM compartment 48 h after transfer. This limited persistence is associated with an increased rate of in vivo apoptosis of TT-specific plasmablasts that have reached the early-life BM and with a significantly lower survival rate of TT-specific plasmablasts cocultured on early-life BM stromal cells compared with adult BM stromal cells. Thus, early-life BM stromal cells fail to provide the molecular signals that support plasmablast survival and differentiation into surviving plasma cells.

Induction of neonatal TH1 and CTL responses by live viral vaccines: a role for replication patterns within antigen presenting cells?

Failure to generate CTL responses in early life has been linked to the preferential maturation of CD4 T cells into TH2 rather than TH1 cells in response to some, but not other, antigenic stimulations. Here, we provide preliminary evidence for the role of the viral replication pattern in the shaping of neonatal cellular responses to live viral vaccines. Neonatal and early life immunization with live attenuated Sendai virus vaccine led to the induction of IgG2a antibodies and cytotoxic responses as efficiently as immunization of adult animals. Similarly, although early life immunization with live attenuated measles virus led to preferential TH2 polarization of T cells compared with adult primed animals, it allowed the induction of CTL responses which had not been observed following immunization with a live recombinant canarypox vector. Thus, conversely to a non-replicating canarypox recombinant vaccine expressing the measles haemagglutonin, viral vaccines with limited but present replication capacity appear capable of activating neonatal antigen presenting cells to trigger TH1 and CTL responses, as recently observed for DNA vaccines.

Specificity of antibodies induced after immunization of mice with the mycobacterial heat shock protein of 65 kD

We have previously shown in mice and monkeys that mycobacterial heat shock proteins (hsp) of 65 and 70 kD exert a strong in vivo helper effect when conjugated to synthetic peptides or bacterial oligosaccharides and given in the absence of any adjuvants. Considering the degree of homology existing in the phylogeny among hsp belonging to the same family, we studied whether antibodies induced in mice with this protocol of immunization with the mycobacterial 65‐kD hsp (hsp65) would cross‐react, and to what extent, with hsp homologues from other origins, notably with the Escherichia coli GroEL protein and with the human homologue (hsp60). The results obtained show that antibodies to the mycobacterial hsp65 cross‐reacted with the E. coli GroEL protein, both in ELISA and Western biol experiments, but not with the human hsp60. In competitive ELISA experiments, the binding of these antibodies to solid‐phase hsp65 was very effectively inhibited by low concentrations of the mycobacterial hsp65; however, for human hsp60. 100 times higher concentrations were required in order to obtain similar patterns of inhibition‐Finally, murine antibodies to the mycobacterial hsp65 always failed to give positive results in Western biol experiments using extracts of murine cells. Taken together, these data suggest that, after immunization of mice with the mycobacterial hsp65 conjugated to peptides or oligosaccharides in the absence of adjuvants, anti‐hsp65 antibodies are induced which cross‐react well with hsp homologues from other prokaryotes (e.g. E. coli GroEL), but which weakly bind the human hsp homologue. These results may have implications for the potential use of microbial hsp molecules in the design of conjugated vaccine constructs.

Environmental and T Cell–Intrinsic Factors Limit the Expansion of Neonatal Follicular T Helper Cells but May Be Circumvented by Specific Adjuvants

Follicular Th (TFH) cells have emerged as a new Th subset providing help to B cells and supporting their differentiation into long-lived plasma cells or memory B cells. Their differentiation had not yet been investigated following neonatal immunization, which elicits delayed and limited germinal center (GC) responses. We demonstrate that neonatal immunization induces CXCR5highPD-1high CD4+ TFH cells that exhibit TFH features (including Batf, Bcl6, c-Maf, ICOS, and IL-21 expression) and are able to migrate into the GCs. However, neonatal TFH cells fail to expand and to acquire a full-blown GC TFH phenotype, as reflected by a higher ratio of GC TFH/non-GC CD4+ T cells in immunized adults than neonates (3.8 × 10−3 versus 2.2 × 10−3, p = 0.01). Following the adoptive transfer of naive adult OT-II CD4+ T cells, OT-II TFH cells expand in the vaccine-draining lymph nodes of immunized adult but not infant recipients, whereas naive 2-wk-old CD4+ OT-II cells failed to expand in adult hosts, reflecting the influence of both environmental and T cell–intrinsic factors. Postponing immunization to later in life increases the number of TFH cells in a stepwise manner, in direct correlation with the numbers of GC B cells and plasma cells elicited. Remarkably, adjuvantation with CpG oligonucleotides markedly increased GC TFH and GC B cell neonatal responses, up to adult levels. To our knowledge, this is the first demonstration that the TFH cell development limits early life GC responses and that adjuvants/delivery systems supporting TFH differentiation may restore adultlike early life GC B cell responses.

Generation of adult‐like antibody avidity profiles after early‐life immunization with protein vaccines

The capacity to induce high‐avidity antibodies following early‐life immunization has long been questioned, and the possibility of inducing such antibodies soon after birth is a recognized goalfor a number of vaccination strategies. Therefore, we assessed the capacity to develop high‐avidity antibodies to peptidic vaccines in 1‐week‐old BALB/c mice. The dynamics of the generation of antibody molecules of increasing avidity were analyzed on circulating serum antibodies and, where feasible, at the single‐cell level on spleen and bone marrow antibody‐secreting cells. Two alum‐adsorbedprotein‐based human vaccines, tetanus toxoid (TT) and pertussis toxin, induced neonatal antibody responses with adult‐like avidity profiles. This was confirmed at the level of spleen and bone marrow ASC. In contrast, immunization with TT‐P30, a 21‐mer synthetic peptide containing a TT‐immunodominant epitope, trinitrophenyl hapten (TNP) conjugated to ovalbumin or TNP conjugated to Ficoll, induced a much lower avidity profile in early life than in adults. These observations indicate that in murine models the avidity maturation of T cell‐dependent antibody responses induced by structurally complex protein vaccines can be fully elicited after early life immunization, as opposed to the maturation of responses induced with short peptides or hapten‐based vaccines.