Andre M. Vale

Long-Term Maintenance of Polysaccharide-Specific Antibodies by IgM-Secreting Cells

Many bacteria-associated polysaccharides induce long-lived Ab responses that protect against pathogenic microorganisms. The maintenance of polysaccharide-specific Ab titers may be due to long-lived plasma cells or ongoing Ag-driven B cell activation due to polysaccharide persistence. BALB/c and VHJ558.3 transgenic mice respond to α1→3-dextran (DEX) by generating a peak anti-DEX response at 7 d, followed by maintenance of serum Ab levels for up to 150 d. Analysis of the cellular response to DEX identified a population of short-lived, cyclophosphamide-sensitive DEX-specific plasmablasts in the spleen, and a quiescent, cyclophosphamide-resistant DEX-specific Ab-secreting population in the bone marrow. BrdU pulse-chase experiments demonstrated the longevity of the DEX-specific Ab-secreting population in the bone marrow. Splenic DEX-specific plasmablasts were located in the red pulp with persisting DEX-associated CD11c+ dendritic cells 90 d after immunization, whereas DEX was not detected in the bone marrow after 28 d. Selective depletion of short-lived DEX-specific plasmablasts and memory B1b B cells using cyclophosphamide and anti-CD20 treatment had a minimal impact on the maintenance of serum anti-DEX Abs. Collectively, these findings demonstrate that the maintenance of serum polysaccharide-specific Abs is the result of continuous Ag-driven formation of short-lived plasmablasts in the spleen and a quiescent population of Ab-secreting cells maintained in the bone marrow for a long duration.

Clinical consequences of defects in B-cell development

Abnormalities in humoral immunity typically reflect a generalized or selective failure of effective B-cell development. The developmental processes can be followed through analysis of cell-surface markers, such as IgM, IgD, CD10, CD19, CD20, CD21, and CD38. Early phases of B-cell development are devoted to the creation of immunoglobulin and testing of B-cell antigen receptor signaling. Failure leads to the absence of B cells and immunoglobulin in the blood from birth. As the developing B cells begin to express a surface B-cell receptor, they become subject to negative and positive selection pressures and increasingly depend on survival signals. Defective signaling can lead to selective or generalized hypogammaglobulinemia, even in the presence of normal numbers of B cells. In the secondary lymphoid organs some B cells enter the splenic marginal zone, where preactivated cells lie ready to rapidly respond to T-independent antigens, such as the polysaccharides that coat some microorganisms. Other cells enter the follicle and, with the aid of cognate follicular T cells, divide to help form a germinal center (GC) after their interaction with antigen. In the GC B cells can undergo the processes of class switching and somatic hypermutation. Failure to properly receive T-cell signals can lead to hyper-IgM syndrome. B cells that leave the GC can develop into memory B cells, short-lived plasma cells, or long-lived plasma cells. The latter ultimately migrate back to the bone marrow, where they can continue to produce protective antigen-specific antibodies for decades.

The Peritoneal Cavity B-2 Antibody Repertoire Appears To Reflect Many of the Same Selective Pressures That Shape the B-1a and B-1b Repertoires

To assess the extent and nature of somatic categorical selection of CDR-3 of the Ig H chain (CDR-H3) content in peritoneal cavity (PerC) B cells, we analyzed the composition of VH7183DJCμ transcripts derived from sorted PerC B-1a, B-1b, and B-2 cells. We divided these sequences into those that contained N nucleotides (N+) and those that did not (N−) and then compared them with sequences cloned from sorted IgM+IgD+ B cells from neonatal liver and both wild-type and TdT-deficient adult bone marrow. We found that the PerC B-1a N− repertoire is enriched for the signatures of CDR-H3 sequences present in neonatal liver and shares many features with the B-1b N− repertoire, whereas the PerC B-1a N+, B-1b N+, and B-2 N+ repertoires are enriched for adult bone marrow sequence signatures. However, we also found several sequence signatures that were not shared with other mature perinatal or adult B cell subsets but were either unique or variably shared between the two or even among all three of the PerC subsets that we examined. These signatures included more sequences lacking N nucleotides in the B-2 population and an increased use of DH reading frame 2, which created CDR-H3s of greater average hydrophobicity. These findings provide support for both ontogenetic origin and shared Ag receptor-influenced selection as the mechanisms that shape the unique composition of the B-1a, B-1b, and B-2 repertoires. The PerC may thus serve as a general reservoir for B cells with Ag binding specificities that are uncommon in other mature compartments.

DH and JH usage in murine fetal liver mirrors that of human fetal liver

In mouse and human, the regulated development of antibody repertoire diversity during ontogeny proceeds in parallel with the development of the ability to generate antibodies to an array of specific antigens. Compared to adult, the human fetal antibody repertoire limits N addition and uses specifically positioned VDJ gene segments more frequently, including V6-1 the most DH-proximal VH, DQ52, the most JH-proximal DH, and JH2, which is DH-proximal. The murine fetal antibody repertoire also limits the incorporation of N nucleotides and uses its most DH proximal VH, VH81X, more frequently. To test whether DH and JH also follow the pattern observed in human, we used the scheme of Hardy to sort B lineage cells from BALB/c fetal and neonatal liver, RT-PCR cloned and sequenced VH7183-containing VDJCμ transcripts, and then assessed VH7183-DH-JH and complementary determining region 3 of the immunoglobulin heavy chain (CDR-H3) content in comparison to the previously studied adult BALB/c mouse repertoire. Due to the deficiency in N nucleotide addition, perinatal CDR-H3s manifested a distinct pattern of amino acid usage and predicted loop structures. As in the case of adult bone marrow, we observed a focusing of CDR-H3 length and CDR-H3 loop hydrophobicity, especially in the transition from the early to late pre-B cell stage, a developmental checkpoint associated with expression of the pre-B cell receptor. However, fetal liver usage of JH-proximal DHQ52 and DH-proximal JH2 was markedly greater than that of adult bone marrow. Thus, the early pattern of DH and JH usage in mouse feta liver mirrors that of human.

Violation of an evolutionarily conserved immunoglobulin diversity gene sequence preference promotes production of dsDNA-specific IgG antibodies.

Variability in the developing antibody repertoire is focused on the third complementarity determining region of the H chain (CDR-H3), which lies at the center of the antigen binding site where it often plays a decisive role in antigen binding. The power of VDJ recombination and N nucleotide addition has led to the common conception that the sequence of CDR-H3 is unrestricted in its variability and random in its composition. Under this view, the immune response is solely controlled by somatic positive and negative clonal selection mechanisms that act on individual B cells to promote production of protective antibodies and prevent the production of self-reactive antibodies. This concept of a repertoire of random antigen binding sites is inconsistent with the observation that diversity (DH) gene segment sequence content by reading frame (RF) is evolutionarily conserved, creating biases in the prevalence and distribution of individual amino acids in CDR-H3. For example, arginine, which is often found in the CDR-H3 of dsDNA binding autoantibodies, is under-represented in the commonly used DH RFs rearranged by deletion, but is a frequent component of rarely used inverted RF1 (iRF1), which is rearranged by inversion. To determine the effect of altering this germline bias in DH gene segment sequence on autoantibody production, we generated mice that by genetic manipulation are forced to utilize an iRF1 sequence encoding two arginines. Over a one year period we collected serial serum samples from these unimmunized, specific pathogen-free mice and found that more than one-fifth of them contained elevated levels of dsDNA-binding IgG, but not IgM; whereas mice with a wild type DH sequence did not. Thus, germline bias against the use of arginine enriched DH sequence helps to reduce the likelihood of producing self-reactive antibodies.

Absence of N addition facilitates B cell development, but impairs immune responses

The programmed, stepwise acquisition of immunocompetence that marks the development of the fetal immune response proceeds during a period when both T cell receptor and immunoglobulin (Ig) repertoires exhibit reduced junctional diversity due to physiologic terminal deoxynucleotidyl transferase (TdT) insufficiency. To test the effect of N addition on humoral responses, we transplanted bone marrow from TdT-deficient (TdT−/−) and wild-type (TdT+/+) BALB/c mice into recombination activation gene 1-deficient BALB/c hosts. Mice transplanted with TdT−/− cells exhibited diminished humoral responses to the T-independent antigens α-1-dextran and (2,4,6-trinitrophenyl) hapten conjugated to AminoEthylCarboxymethyl-FICOLL, to the T-dependent antigens NP19CGG and hen egg lysozyme, and to Enterobacter cloacae, a commensal bacteria that can become an opportunistic pathogen in immature and immunocompromised hosts. An exception to this pattern of reduction was the T-independent anti-phosphorylcholine response to Streptococcus pneumoniae, which is normally dominated by the N-deficient T15 idiotype. Most of the humoral immune responses in the recipients of TdT−/− bone marrow were impaired, yet population of the blood with B and T cells occurred more rapidly. To further test the effect of N-deficiency on B cell and T cell population growth, transplanted TdT-sufficient and -deficient BALB/c IgMa and congenic TdT-sufficient CB17 IgMb bone marrow were placed in competition. TdT−/− cells demonstrated an advantage in populating the bone marrow, the spleen, and the peritoneal cavity. TdT deficiency, which characterizes fetal lymphocytes, thus appears to facilitate filling both central and peripheral lymphoid compartments, but at the cost of altered responses to a broad set of antigens.

The CDR-H3 Repertoire from TdT-Deficient Adult Bone Marrow Is a Close, but Not Exact, Homologue of the CDR-H3 Repertoire from Perinatal Liver

Compared with adult bone marrow (BM), the composition of the perinatal liver CDR-3 of the Ig H chain (CDR-H3) repertoire is marked by a paucity of N nucleotides and by enrichment for use of JH proximal DQ52 and DH proximal VH and JH gene segments. To test the extent to which these differences reflect limited perinatal TdT activity versus differences in the fetal/adult environment, we used the Hardy scheme to sort fractions B–F B lineage cells from TdT-deficient BALB/c adult BM. VH7183-containing VDJCμ transcripts from these cells were amplified, cloned, sequenced, and compared with transcripts from wild-type perinatal liver and adult BM. The pattern of VHDJH usage in TdT-deficient BM largely matched that of TdT-sufficient adult cells. What minor differences were detected in the pro-B cell stage tended to diminish with B cell maturation, suggesting strong environmental or Ag-driven pressure to achieve a specific range of VHDJH usage regardless of the extent of N nucleotide addition. However, although the patterns of VHDJH usage in the TdT-deficient B lineage cells paralleled that of wild-type adult cells, the length distribution, global amino acid composition, and charge distribution of the CDR-H3 repertoire proved to be a close, although not exact, homologue of the CDR-H3 repertoire first expressed by late pre-B cells in the TdT-insufficient perinatal liver. Thus, although differing in VH content, TdT-deficient mice appear to represent a good, although not perfect, model for testing the role of perinatal CDR-H3 limitations on late B cell development and Ab responses.

GENETIC CONTROL OF THE B CELL RESPONSE TO LPS: OPPOSING EFFECTS IN PERITONEAL VERSUS SPLENIC B CELL POPULATIONS

Lipopolysaccharide (LPS) from gram-negative bacteria activates B cells, enabling them to proliferate and differentiate into plasma cells. This response is critically dependent on the expression of TLR4; but other genes, such as RP105 and MHC class II, have also been shown to contribute to B cell LPS response. Here, we have evaluated the role of genetic control of the B cell response to LPS at the single cell level. We compared the response to LPS of peritoneal cavity (PEC) and splenic B cells on the BALB/c genetic background (LPS-low responder) to those on the C57BL/6J background (LPS-high responder) and their F1 progeny (CB6F1). Both PEC and splenic B cells from B6 exhibited 100% clonal growth in the presence of LPS; whereas, BALB/c PEC and splenic B cells achieved only 50% and 23% clonal growth, respectively. Adding CpG to the LPS stimulus pushed PEC B cell clonal growth in the low responder strain BALB/c up to 90%, showing that the nonresponse to LPS is a specific effect. Surprisingly, PEC B cells on the F1 background behaved as high responders, while splenic B cells behaved as low responders to LPS. The data presented here reveals a previous unsuspected behavior in the genetic control of the B cell response to LPS with an opposing impact in splenic versus peritoneal cavity B cells. These results suggest the existence of an, as yet, unidentified genetic factor exclusively expressed by coelomic B cells that contributes to the control of the LPS signaling pathway in the B lymphocyte.

The Global Self-Reactivity Profile of the Natural Antibody Repertoire Is Largely Independent of Germline DH Sequence

Natural antibodies (NAbs) are produced in the absence of exogenous antigenic stimulation and circulate in the blood of normal, healthy individuals. These antibodies have been shown to provide one of the first lines of defense against both bacterial and viral pathogens. Conservation of the NAb repertoire reactivity profile is observed both within and across species. One view holds that this conservation of NAb self-reactivities reflects the use of germline antibody sequence, whereas the opposing view holds that the self-reactivities reflect selection driven by key conserved self-antigens. In mice, B-1a B cells are a major source of NAbs. A significant fraction of the B-1a antibody repertoire is devoid of N nucleotides in H chain complementarity determining region 3 (CDR-H3) and, thus, completely germline encoded. To test the role of germline DH sequence on the self-reactivity profile of the NAb repertoire, we examined the composition and self-antigen specificity of NAbs produced by a panel of DH gene-targeted BALB/c mice, each strain of which expresses a polyclonal, altered CDR-H3 repertoire that differs from the wild-type norm. We found that in most cases the same key self-antigens were recognized by the NAbs created by each DH-altered strain. The differences in reactivity appeared to represent the genetic signature of the NAb repertoire of each mouse strain. These findings suggest that although germline CDR-H3 sequence may facilitate the production of certain NAbs, a core set of self-antigens are likely the main force driving the selection of Nab self-specificities.

A rapid and quantitative method for the evaluation of V gene usage, specificities and the clonal size of B cell repertoires.

The quantitative simultaneous description of both variable region gene usage and antigen specificity of immunoglobulin repertoires is a major goal in immunology. Current quantitative assays are labor intensive and depend on extensive gene expression cloning prior to screening for antigen specificity. Here we describe an alternative method based on high efficiency single B cell cultures coupled with RT-PCR that can be used for rapid characterization of immunoglobulin gene segment usage, clonal size and antigen specificity. This simplified approach should facilitate the study of antibody repertoires expressed by defined B cell subpopulations, the analysis of immune responses to self and nonself-antigens, the development and screening of synthetic antibodies and the accelerated study and screening of neutralizing antibodies to pathogenic threats.