Primary Ab Responses Research Articles

Characterization of the primary antibody response to Plasmodium falciparum antigens in infants living in a malaria-endemic area

BackgroundThe primary antibody (Ab) response to Plasmodium falciparum is a critical step in developing immunity to malaria. Information on the initial Ab responses of babies in malaria-endemic areas is incomplete, in part, because babies receive maternal IgG via transplacental-transfer and usually become infected before maternal IgG wanes. The study aimed to identify the primary IgM and IgG Ab responses to malarial antigens in Cameroonian babies.MethodsInfants (n = 70) living in a high malaria transmission area were followed from birth throughout the first year of life (mean 341 ± 42 days, an average of 8.5 time points per infant). Malaria infection was assessed by microscopy and PCR, and IgM and IgG antibodies (Abs) were measured using a multiplex immunoassay to AMA1, EBA-175, MSP1-42, MSP2, MSP3, RESA, LSA1, and CSP.ResultsThe half-life of maternal IgG varied among the antigens, ranging from 0.7 to 2.5 months. The first infection of 41% of the babies was sub-microscopic and only 11 to 44% of the babies produced IgM to the above antigens; however, when the first infection was detected by microscopy, 59–82% of the infants made IgM Abs to the antigens. Infants were able to produce IgM even when maternal IgG was present, suggesting maternal Abs did not suppress the baby’s initial Ab response. Using longitudinal regression models that incorporated time-varying covariates, infants were found to produce IgG Ab to only AMA-1 when the first infection was sub-microscopic, but they produced IgG Abs to MSP1-42 (3D7, FVO), AMA1 (3D7, FVO) MSP2-FC27, MSP3, RESA, and LSA1, but not MSP 2-3D7, EBA-175, and CSP during their first slide-positive infection. Notably, the primary and secondary IgG responses were short-lived with little evidence of boosting.ConclusionsThe primary Ab response of babies who had maternal IgG was similar to that reported for primary infections in malaria-naïve adults.

FliC's Hypervariable D3 Domain Is Required for Robust Anti-Flagellin Primary Antibody Responses.

Bacterial flagellin is a well-known agonist of the innate immune system that induces proinflammatory responses through the TLR5 and Naip5/6 recognition pathways. Several clinical trials investigating flagellin fusion proteins have demonstrated promising results for inducing protective immunity toward influenza virus, which has been largely attributed to flagellin's ability to activate TLR5. Our laboratory previously demonstrated that the Salmonella enterica serovar Typhimurium flagellin protein, FliC, induces Ab responses in mice through a third pathway that is independent of TLR5, Casp1/11, and MyD88. In this study, we further define the structural features of FliC that contribute to this unknown third pathway. By destroying the Naip5/6 and TLR5 recognition sites, we demonstrate that neither were required for the TLR5-, inflammasome- and MyD88-independent Ab responses toward FliC. In contrast, deletion of FliC's D3 or D0/D1 domains eliminated primary anti-flagellin Ab responses. For optimal primary and secondary anti-flagellin Ab responses we show that TLR5, inflammasome recognition, and the D3 domain of FliC are essential for flagellin's robust immunogenicity. Our data demonstrate that the D3 domain of FliC influences immunogenicity independent of the known innate recognition sites in the D0/D1 domains to augment Ab production. Our results suggest full-length FliC is critical for optimal immunogenicity and Ab responses in flagellin-based vaccines.

ZBTB32 Restricts the Duration of Memory B Cell Recall Responses.

Memory B cell responses are more rapid and of greater magnitude than are primary Ab responses. The mechanisms by which these secondary responses are eventually attenuated remain unknown. We demonstrate that the transcription factor ZBTB32 limits the rapidity and duration of Ab recall responses. ZBTB32 is highly expressed by mouse and human memory B cells but not by their naive counterparts. Zbtb32(-/-) mice mount normal primary Ab responses to T-dependent Ags. However, Zbtb32(-/-) memory B cell-mediated recall responses occur more rapidly and persist longer than do control responses. Microarray analyses demonstrate that Zbtb32(-/-) secondary bone marrow plasma cells display elevated expression of genes that promote cell cycle progression and mitochondrial function relative to wild-type controls. BrdU labeling and adoptive transfer experiments confirm more rapid production and a cell-intrinsic survival advantage of Zbtb32(-/-) secondary plasma cells relative to wild-type counterparts. ZBTB32 is therefore a novel negative regulator of Ab recall responses.

B Lymphocyte-Specific Loss of Ric-8A Results in a Gα Protein Deficit and Severe Humoral Immunodeficiency.

Resistance to inhibitors of cholinesterase 8A (Ric-8A) is a highly evolutionarily conserved cytosolic protein initially identified in Caenorhabditis elegans, where it was assigned a regulatory role in asymmetric cell divisions. It functions as a guanine nucleotide exchange factor for Gαi, Gαq, and Gα12/13 and as a molecular chaperone required for the initial association of nascent Gα subunits with cellular membranes in embryonic stem cell lines. To test its role in hematopoiesis and B lymphocytes specifically, we generated ric8 (fl/fl) vav1-cre and ric8 (fl/fl) mb1-cre mice. The major hematopoietic cell lineages developed in the ric8 (fl/fl) vav1-cre mice, notwithstanding severe reduction in Gαi2/3, Gαq, and Gα13 proteins. B lymphocyte-specific loss of Ric-8A did not compromise bone marrow B lymphopoiesis, but splenic marginal zone B cell development failed, and B cells underpopulated lymphoid organs. The ric8 (fl/fl) mb1-cre B cells exhibited poor responses to chemokines, abnormal trafficking, improper in situ positioning, and loss of polarity components during B cell differentiation. The ric8 (fl/fl) mb1-cre mice had a severely disrupted lymphoid architecture and poor primary and secondary Ab responses. In B lymphocytes, Ric-8A is essential for normal Gα protein levels and is required for B cell differentiation, trafficking, and Ab responses.

Staphylococcal Enterotoxin B (SEB) Induces Memory CD4 T Cell Anergy in vivo and Impairs Recall Immunity to Unrelated Antigens.

IntroductionNaïve and memory T cells can utilize unique regulatory pathways to promote protection but prevent self-reactivity. A bacterial superantigen SEB exploits unique TCR proximal signaling processes in memory CD4 T cells to induce clonal anergy. The aim of this study was to determine if SEB could antagonize memory CD4 T cells in vivo and whether there would be consequences on recall immune responses. We evaluated Ab responses to a T-dependent antigen as a measurement of memory T cell helper function.MethodBALB/c mice were primed with TNP-RGG to elicit memory B cells and also immunized with an ovalbumin peptide to elicit memory helper T cells. Another group of TNP-RGG immunized mice were used as adoptive transfer recipients of exogenous DO11.10 memory T cells. Mice were challenged with TNP-OVA with or without prior administration of SEB. B cells secreting IgM or IgG TNP-specific Ab were enumerated by ELISPOT as indicators of primary versus secondary humoral immunity.ResultsComparing the SEB and non-SEB-treated groups, the SEB-treated group failed to produce TNP-specific IgG in response to challenge with TNP-OVA, even if they were previously immunized with OVA. All groups produced IgM, indicating that the primary Ab responses and naïve helper T cells were not impacted by SEB. SEB had no negative impact when DO11.10 × Fyn−/− memory T cells were used as donor cells.ConclusionThe present study indicated that SEB selectively targeted memory CD4 T cells in vivo and prevented helper function. Consequently, recall humoral immunity was lost. The data are most consistent with in vivo T cell anergy as opposed to indirect suppression as elimination of Fyn kinase restored helper function. These data suggest that bacterial superantigens can impair post-vaccination memory cell responses to unrelated antigens via their ability to target Vb families and antagonize memory cell activation.

Antibody response specific to the capsular polysaccharide is impaired in Streptococcus suis serotype 2-infected animals.

Streptococcus suis serotype 2 is an extracellular encapsulated bacterium that causes severe septicemia and meningitis in swine and humans. Albeit crucial in the fight against encapsulated bacteria, the nature of the capsular polysaccharide (CPS)-specific antibody (Ab) response during S. suis type 2 infection is unknown. We compared for the first time the features of CPS-specific versus protein-specific Ab responses during experimental infections with live virulent S. suis type 2 in mice. The primary protein-specific Ab response was dominated by both type 1 and 2 IgG subclasses, whereas IgM titers were more modest. The secondary protein-specific Ab response showed all of the features of a memory response with faster kinetics and boosted the titers of all Ig isotypes. In contrast, the primary CPS-specific Ab response was either inexistent or had titers only slightly higher than those in noninfected animals and was essentially composed of IgM. A poor CPS-specific memory response was observed, with only a moderate boost in IgM titers and no IgG. Both protein- and CPS-specific Ab responses were Toll-like receptor 2 independent. By using S. suis type 2 strains of European or North American origin, the poor CPS-specific Ab response was demonstrated to be independent of the genotypic/phenotypic diversity of the strain within serotype 2. Finally, the CPS-specific Ab response was also impaired and lacked isotype switching in S. suis-infected pigs, the natural host of the bacterium. The better resistance of preinfected animals to reinfection with the same strain of S. suis type 2 might thus more likely be related to the development of a protein rather than CPS Ab response.

Modulation of Antigen Presentation and B Cell Receptor Signaling in B Cells of Beige Mice

Binding of Ag by B cells leads to signal transduction downstream of the BCR and to delivery of the internalized Ag-BCR complex to lysosomes where the Ag is processed and presented on MHC class II molecules. T cells that recognize the peptide-MHC complexes provide cognate help to B cells in the form of costimulatory signals and cytokines. Recruitment of T cell help shapes the Ab response by facilitating isotype switching and somatic hypermutation, and promoting the generation of memory cells and long-lived plasma cells. We have used the beige (Bg) mouse, which is deficient in endosome biogenesis, to evaluate the effect of potentially altered Ag presentation in shaping the humoral response. We show that movement of the endocytosed Ag-BCR complex to lysosomes is delayed in Bg B cells and leads to relatively poorer stimulation of Ag-specific T cells. Nevertheless, this does not affect Bg B cell activation or proliferation when competing with wild-type B cells for limiting T cell help in vitro. Interestingly, Bg B cells show more prolonged phosphorylation of signaling intermediates after BCR ligation and proliferate better to low levels of BCR cross-linking. Primary Ab responses are similar in both strains, but memory responses and plasma cell frequencies in bone marrow are higher in Bg mice. Further, Bg B cells mount a higher primary Ab response when competing with wild-type cells in vivo. Thus, the intensity and duration of BCR signaling may play a more important part in shaping B cell responses than early Ag presentation for T cell help.

Heligmosomoides polygyrus Elicits a Dominant Nonprotective Antibody Response Directed against Restricted Glycan and Peptide Epitopes

Heligmosomoides polygyrus is a widely used gastrointestinal helminth model of long-term chronic infection in mice, which has not been well-characterized at the antigenic level. We now identify the major targets of the murine primary Ab response as a subset of the secreted products in H. polygyrus excretory-secretory (HES) Ag. An immunodominant epitope is an O-linked glycan (named glycan A) carried on three highly expressed HES glycoproteins (venom allergen Ancylostoma-secreted protein-like [VAL]-1, -2, and -5), which stimulates only IgM Abs, is exposed on the adult worm surface, and is poorly represented in somatic parasite extracts. A second carbohydrate epitope (glycan B), present on both a non-protein high molecular mass component and a 65-kDa molecule, is widely distributed in adult somatic tissues. Whereas the high molecular mass component and 65-kDa molecules bear phosphorylcholine, the glycan B epitope itself is not phosphorylcholine. Class-switched IgG1 Abs are found to glycan B, but the dominant primary IgG1 response is to the polypeptides of VAL proteins, including also VAL-3 and VAL-4. Secondary Ab responses include the same specificities while also recognizing VAL-7. Although vaccination with HES conferred complete protection against challenge H. polygyrus infection, mAbs raised against each of the glycan epitopes and against VAL-1, VAL-2, and VAL-4 proteins were unable to do so, even though these specificities (with the exception of VAL-2) are also secreted by tissue-phase L4 larvae. The primary immune response in susceptible mice is, therefore, dominated by nonprotective Abs against a small subset of antigenic epitopes, raising the possibility that these act as decoy specificities that generate ineffective humoral immunity.

Requirement for complement in antibody responses is not explained by the classic pathway activator IgM

Animals lacking complement factors C1q, C2, C3, or C4 have severely impaired Ab responses, suggesting a major role for the classic pathway. The classic pathway is primarily initiated by antigen-Ab complexes. Therefore, its role for primary Ab responses seems paradoxical because only low amounts of specific Abs are present in naive animals. A possible explanation could be that the classic pathway is initiated by IgM from naive mice, binding with sufficient avidity to the antigen. To test this hypothesis, a knock-in mouse strain, Cμ13, with a point mutation in the gene encoding the third constant domain of the μ-heavy chain was constructed. These mice produce IgM in which proline in position 436 is substituted with serine, a mutation previously shown to abrogate the ability of mouse IgM to activate complement. Unexpectedly, the Ab response to sheep erythrocytes and keyhole limpet hemocyanin in Cμ13 mice was similar to that in WT mice. Thus, although secreted IgM and the classic pathway activation are both required for the normal primary Ab response, this does not require that IgM activate C. This led us to test Ab responses in animals lacking one of three other endogenous activators of the classic pathway: specific intracellular adhesion molecule-grabbing nonintegrin R1, serum amyloid P component, and C-reactive protein. Ab responses were also normal in these animals.

Characterizing the primary B cell antibody response to HIV (53.18)

Abstract Most individuals infected with the human immunodeficiency virus (HIV) make neutralizing antibodies (nAbs) to the virus, but this response is delayed and consequently not protective. Some nAbs that have been characterized are described as polyreactive and slightly autoreactive, two attributes of Abs produced by marginal zone (MZ) B cells. Thus, B cells capable of producing nAbs early in infection may not participate, possibly because of tolerance mechanisms. To directly examine the components of the primary Ab response to the HIV envelope protein gp120, we use a mouse model and HIV viral-like particles (VLPs). Our studies focus on the quantity and quality of the anti-HIV responses from different B cell subsets. We have found that naïve follicular (FO) and MZ B cells are both capable of producing anti-gp120 IgM in culture. Additionally, we have examined the VDJ rearrangements of gp120-reactive Abs from different populations in both the naïve and VLP-challenged repertoire. So far, it seems that there may be a preferred VDJ recombination event that is over-represented in the MZ B cell compartment. This VDJ rearrangement has not yet been found in responding cells of VLP-immunized mice, indicating that MZ B cells may not be participating in the VLP response. Abs from these subsets are being further characterized with HIV-neutralization studies. It is expected that the results from these experiments will prove informative for the development of a protective HIV vaccine.

Dendritic cell-B cell interactions in the marginal zone drive extrafollicular antibody responses. (61.4)

Abstract Targeting Ag directly to dendritic cell (DC) subsets through the use of monoclonal antibodies (mAb) is increasingly being employed as a means to generate CD4 and CD8 T cell responses. However, the ability of individual DC subsets to raise primary Ab responses following Ag targeting is not well characterized. Using the hapten 4-hydroxy-3-nitrophenyl (NP) coupled to mAbs that recognize DC-specific DEC205 or DCIR2, we found that MZ-associated DCIR2+ DCs, but not DEC205+ DCs, were able to induce robust T cell- and CD40-dependent anti-NP Ab responses following injection of soluble Ag. Anti-NP Ab responses were restricted to the IgG1 sub-class but failed to either persist over time or undergo affinity maturation, despite the addition of TLR or RLR agonists. Using adoptive transfers with NP-specific B cells, we show that the unique ability of DCIR2+ DCs to induce Ab responses was associated with rapid, MHC class II-independent activation of Ag-specific B cells that occurred specifically upon Ag delivery to DCIR2. Importantly, we found that NP-specific B cells accumulated in MZ-associated bridging channels in close association with CD11c+ cells as early as 24hr following Ag delivery to DCIR2+ DCs. Together, these results reveal a previously unrecognized role for DC-B cell interactions in splenic bridging channels, and provide a novel mechanism for the initiation of T-dependent extrafollicular Ab responses.

Selective induction of robust antibody responses following antigen targeting to dendritic cell subsets (90.34)

Abstract Dendritic cells (DCs) are essential for the initiation and regulation of immune responses. CD8α+ DCs in the mouse spleen express DEC-205 (CD205), while many splenic CD8α- DCs express DC-inhibitory receptor-2 (DCIR2/33D1). Delivering antigen (Ag) via mAbs to either DEC-205 or DCIR2 was shown by other groups to preferentially induce CD8+ and CD4+ T cell responses, respectively. However, the ability of these two DC subsets to induce primary Ab responses is not well defined. Using the conventional hapten (4-hydroxy-3-nitrophenyl)acetyl (NP) coupled to mAbs that recognize DEC-205 or DCIR2, we demonstrate that DCIR2+ DCs, but not DEC-205+ DCs, are able to induce robust anti-NP Ab responses following intravenous injection of soluble Ag. These Ab responses were restricted to the IgG1 subclass with little to no IgM or IgG2a production. Surprisingly, the DCIR2-induced Ab response did not require the addition of conventional adjuvants. These responses were both TCR and CD40-dependent, indicating that T-B cell collaboration is required. Finally, we found that CD22 expression by B cells was also required for IgG1 anti-NP responses, suggesting that DC-B cell interactions may be important during early B cell priming via this route of administration. Together these results demonstrate that, similar to the selective priming of T cells by these DC subsets, DCs are also specially equipped to selectively prime B cells and Ab responses. This project was supported in part by NAIAD training grant T32AI007411 and by NIH grant AI52203 and does not necessarily represent the official views of the NAIAD or the NIH.

Effects of dietary chromium supplementation on glucose tolerance and primary antibody response against peste des petits ruminants in dwarf Bengal goats (Capra hircus)

An experiment was conducted to exploit the immune-modulation effect of trivalent chromium (Cr) for augmenting immunity against peste des petits ruminants (PPR) in dwarf black Bengal goats (Capra hircus). The goats (n = 6 per treatment group) either received a basal diet devoid of supplemental Cr (control) or were supplemented with 0.5 mg Cr as chromic chloride (+Cr) for 60 days. Live weight gain and feed conversion efficiency improved (P < 0.05) in the +Cr dietary group. Supplemental Cr did not affect the total number of monocytes, eosinophils and basophils (P > 0.1) although total leukocytes increased (P < 0.05) and the ratio of neutrophils to lymphocytes narrowed down (P < 0.05) in the +Cr dietary group. The effect of Cr supplementation on variables of the intravenous glucose tolerance test (IVGTT) was inconclusive. Compared to the control group, the basal concentration of glucose (P < 0.05) and insulin (P < 0.001) was higher, clearance rate was slower (P < 0.05) and serum half-life was greater (P < 0.05) in the +Cr dietary group during the IVGTT. Over the duration of the experiment, serum concentration of insulin increased (P < 0.001) and that of cortisol decreased (P < 0.01) in the +Cr group, which also showed a relatively higher primary antibody (Ab) response against PPR on days 10 (P < 0.01) and 20 (P < 0.05) post-vaccination. In conclusion, the experiment indicated that supplementation of Cr as CrCl3, in the diet of non-stressed goats, may improve primary Ab response against PPR and help confer an augmented immunity to the disease besides promoting growth and feed conversion.

Deregulation of c-Myc Confers Distinct Survival Requirements for Memory B Cells, Plasma Cells, and Their Progenitors

Deregulation of the c-Myc oncogene is tightly associated with human and murine plasma cell (PC) neoplasms. Through the analysis of Ag-specific B cell responses in mice where Myc is targeted to the Igh Calpha locus, we show here that c-Myc dramatically impairs the primary and secondary Ab response. This impairment is differentiation stage specific, since germinal center B cell formation, affinity maturation, and class switch recombination were intact. Examination of PC viability revealed that c-Myc triggered apoptosis only upon final maturation when Ab is secreted and is resistant to the survival factor BAFF (B cell-activating factor belonging to the TNF family). In contrast, PC precursors (PC(pre)) that ultimately give rise to mature PCs survived normally and vigorously expanded with BAFF signaling. We further show that c-Myc also facilitates the apoptosis of memory B cells. Thus, Calpha-Myc controls both cellular arms of long-lived B cell immunity than previously anticipated. Only when deregulation of c-Myc was combined with enforced Bcl-x(L) expression were mature PCs able to survive in response to BAFF. These data indicate that the survival requirements for tumor-susceptible PC(pre) and PCs are distinct and that tumor progression likely develops as PC(pre) transition to functional PCs when apoptotic pathways such as members of the Bcl-2 family are disabled.

Decreased Specific Antibody Responses to α-Gal-Conjugated Antigen in Animals with Preexisting High Levels of Natural Antibodies Binding α-Gal Residues

High levels of natural antibodies (NAb) binding the alpha-Gal residue (Galalpha1-3Galbeta1-4GlcNAc) are found in poultry (and humans), which is probably reflected by high levels of natural agglutinating antibodies (Ab) to rabbit red blood cells (RRBC) in plasma from chickens (and humans). Recently, it was shown that alpha-Gal conjugation of proteins induced higher antiprotein Ab responses in alpha-Gal knockout mice, suggesting immune-enhancing features of preexisting Ab binding carbohydrate-protein conjugates. We challenged chickens s.c. with either alpha-Gal-conjugated human serum albumin (HuSA), beta-Gal-conjugated HuSA, or unconjugated ("native") HuSA, respectively, and measured primary and secondary Ab responses to HuSA, including isotype IgM and IgG responses, and cellular immune responses in vitro (lymphoproliferation) to HuSA or concanavalin A. alpha-Gal conjugation, but not beta-Gal conjugation, of HuSA resulted in significantly decreased primary and secondary Ab responses to HuSA, especially IgG isotype responses, as compared with Ab responses to native HuSA. Lymphoproliferation in vitro was also decreased, although not significantly, in birds challenged with alpha-Gal-conjugated HuSA. High levels of agglutinating Ab levels to RRBC and NAb binding porcine thyroglobulin were detected in all birds, as was true for (natural) Ab levels binding alpha-Gal-conjugated HuSA before immunization, whereas low levels of preexisting (natural) antibodies directed to native HuSA were present in plasma before immunization. Levels of RRBC agglutinins and Ab binding thyroglobulin were not affected by immunization with HuSA, alpha-Gal-conjugated HuSA, or beta-Gal-conjugated HuSA. Our data confirm the presence of high levels of (preexisting) NAb in the plasma of chickens directed to the alpha-Gal residue. The decreased responsiveness to alpha-Gal-bearing antigens in the current study shows that, in addition to immune-enhancing features, NAb may also have suppressive effects on specific immune responses, which substantiates the regulatory role of innate immunity (NAb) in mounting specific immune responses.

CD36 Is Differentially Expressed on B Cell Subsets during Development and in Responses to Antigen

Of a number of mAbs made by immunization with sort-purified marginal zone (MZ) B cells, one was shown to recognize the mouse scavenger receptor CD36. Although CD36 is expressed by most resting MZ B cells and not by follicular and B1 B cells, it is rapidly induced on follicular B cells in vitro following TLR and CD40 stimulation. In response to T-independent and T-dependent Ag challenge, we found that CD36 was expressed on IgM+ plasma cells, but down-regulated on isotype-switched plasma cells in vivo. Although development, localization, and phenotype of MZ B cells in CD36-/- mice appeared normal, there was a minor block in the transitional stages of mature B cell development. In both primary and secondary Ab responses to heat-killed Streptococcus pneumoniae (R36A strain), both phosphoryl choline (PC)-specific IgM and IgG levels in CD36-/- mice were slightly reduced compared with wild-type mice. In addition, mice deficient in both TLR2 and CD36 produced significantly reduced levels of anti-PC IgG titers than those of single gene-deficient mice, suggesting that they may cooperate in an anti-PC Ab response. Collectively, these results show that CD36 does not affect the development of B cells, but modulates both primary and secondary anti-PC Ab responses during S. pneumoniae infection similarly to TLR2.

Maintenance of Long-Lived Plasma Cells and Serological Memory Despite Mature and Memory B Cell Depletion during CD20 Immunotherapy in Mice

CD20 mAb-mediated B cell depletion is an effective treatment for B cell malignancies and some autoimmune diseases. However, the full effects of B cell depletion on natural, primary, and secondary Ab responses and the maintenance of Ag-specific serum Ig levels are largely unknown. The relationship between memory B cells, long-lived plasma cells, and long-lived humoral immunity also remains controversial. To address the roles of B cell subsets in the longevity of humoral responses, mature B cells were depleted in mice using CD20 mAb. Peritoneal B cell depletion reduced natural and Ag-induced IgM responses. Otherwise, CD20+ B cell depletion prevented humoral immune responses and class switching and depleted existing and adoptively transferred B cell memory. Nonetheless, B cell depletion did not affect serum Ig levels, Ag-specific Ab titers, or bone marrow Ab-secreting plasma cell numbers. Coblockade of LFA-1 and VLA-4 adhesion molecules temporarily depleted long-lived plasma cells from the bone marrow. CD20+ B cell depletion plus LFA-1/VLA-4 mAb treatment significantly prolonged Ag-specific plasma cell depletion from the bone marrow, with a significant decrease in Ag-specific serum IgG. Collectively, these results support previous claims that bone marrow plasma cells are intrinsically long-lived. Furthermore, these studies now demonstrate that mature and memory B cells are not required for maintaining bone marrow plasma cell numbers, but are required for repopulation of plasma cell-deficient bone marrow. Thereby, depleting mature and memory B cells does not have a dramatic negative effect on preexisting Ab levels.

NAD+ Released during Inflammation Participates in T Cell Homeostasis by Inducing ART2-Mediated Death of Naive T Cells In Vivo

Mono ADP-ribosyltransferase 2 (ART2) is an ectoenzyme expressed on mouse T lymphocytes, which catalyze the transfer of ADP-ribose groups from NAD(+) onto several target proteins. In vitro, ADP-ribosylation by ART2 activates the P2X7 ATP receptor and is responsible for NAD(+)-induced T cell death (NICD). Yet, the origin of extracellular NAD(+) and the role of NICD in vivo remain elusive. In a model of acute inflammation induced by polyacrylamide beads, we demonstrate release of NAD(+) into exudates during the early phase of the inflammatory response. This leads to T cell depletion in the draining lymph nodes from wild-type and, more severely, from mice lacking the CD38 NAD(+) glycohydrolase, whereas no effect is observed in ART2-deficient animals. Intravenous injection of NAD(+) used to exacerbate NICD in vivo results in fast and dramatic ART2- and P2X7-dependent depletion of CD4+ and CD8+ T lymphocytes, which can affect up to 80% of peripheral T cells in CD38(-/-) mice. This affects mainly naive T cells as most cells surviving in vivo NAD+ treatment exhibit the phenotype of recently activated/memory cells. Consistently, treatment with NAD(+) abolishes primary Ab response to a T-dependent Ag in NICD-susceptible CD38(-/-) mice but has no effect on the secondary response when given several days after priming. Unexpectedly NAD+ treatment improves the response in their wild-type BALB/c counterparts. We propose that NAD(+) released during early inflammation facilitates the expansion of primed T cells, through ART2-driven death of resting cells, thus contributing to the dynamic regulation of T cell homeostasis.

Induction of human primary antibody responses in vitro by ovalbumin (OVA) or anthrax (rPA) on dendritic cells (DCs) and follicular dendritic cells (FDCs) in temporally controlled germinal center reactions (GCRs) (47.19)

Abstract Induction of potent primary human Ab responses entirely in vitro is challenging, although such in vitro responses could be helpful in assessing vaccines and therapeutics without jeopardizing human health. Given that DCs prime T cells and that FDCs promote B cell responses, we hypothesized that primary Ab responses could be induced in temporally controlled GCRs in vitro. To test this, monocyte derived-DCs were given OVA or rPA for processing, matured with LPS, and CD4+ T cells were added for priming. Ten days later, naïve B cells and OVA or rPA bearing FDCs were added and Ab production was subsequently assayed by ELISA. The CD4+ T cells clustered with DCs through the 10 days of priming and fused with FDC-B cell clusters when GCRs were induced. IgM anti-OVA levels were 79±15 ng/ml 7 days after adding B cells and declined to 2.4±0.5 ng/ml at day 14. In contrast, IgG anti-OVA rose from 9±1.7 at day 7 to 25±6.4 ng/ml at day 14 and the IgG1:IgG2 ratio was 1:3. Similarly, IgM anti-rPA levels were 10 ng/ml by just 5 days then IgM production switched to IgG. IgG anti-rPA rose from 3 ng/ml at day 5 to 9 ng/ml by day 12. In short, Ag specific primary human Ab responses, including Ig class switching from specific IgM to IgG, can be generated in GCRs entirely in vitro. Thus, potential vaccines can be rapidly and safely assessed using the human immune system. (Supported and cleared by DARPA on 12/20/2006 - Approved for Public Release, Distribution Unlimited)

A Critical Role for Complement C3d and the B Cell Coreceptor (CD19/CD21) Complex in the Initiation of Inflammatory Arthritis

Complement C3 cleavage products mediate the recognition and clearance of toxic or infectious agents. In addition, binding of the C3d fragment to Ag promotes B lymphocyte activation through coengagment of the BCR and complement receptor 2 (CD21). Signal augmentation is thought to be achieved through enhanced recruitment and activation of CD21-associated CD19. In this study we show, using the DBA/1 collagen-induced arthritis (CIA) model, that conjugation of C3d to heterologous type II collagen is sufficient to cause disease in the absence of the mycobacterial components of CFA. Transient depletion of C3 during the inductive phase of CIA delays and lessens the severity of disease, and DBA/1 mice deficient for coreceptor components CD19 or CD21 are not susceptible to CIA. Adoptive transfer experiments revealed that CD21 expression on either B cells or follicular dendritic cells is sufficient to acquire disease susceptibility. Although CD19(-/-) and CD21(-/-) mice produce primary Ab responses to heterologous and autologous type II collagen, they are impaired in the ability to activate T cells, form germinal centers, and produce secondary autoantibody responses. These findings indicate that binding of C3d to self-Ags can promote autoimmunity through enhanced Ag retention and presentation by follicular dendritic cells and B cells, respectively.