Combinatorial library cloning of human antibodies to Streptococcus pneumoniae capsular polysaccharides: variable region primary structures and evidence for somatic mutation of Fab fragments specific for capsular serotypes 6B, 14, and 23F.

Antibodies specific for capsular polysaccharides play a central role in immunity to encapsulated Streptococcus pneumoniae, but little is known about their genetics or the variable (V) region polymorphisms that affect their protective function. To begin to address these issues, we used combinatorial library cloning to isolate pneumococcal polysaccharide (PPS)-specific Fab fragments from two vaccinated adults. We determined complete V region primary structures and performed antigen binding analyses of seven Fab fragments specific for PPS serotype 6B, 14, or 23F. Fabs were of the immunoglobulin G2 or A isotype. Several V(H)III gene segments (HV 3-7, 3-15, 3-23, and 3-11) were identified. V(L) regions were encoded by several kappa genes (KV 4-1, 3-15, 2-24, and 2D-29) and a lambda gene (LV 1-51). Deviation of the V(H) and V(L) regions from their assigned germ line counterparts indicated that they were somatically mutated. Fabs of the same serotype specificity isolated from a single individual differed in affinity, and these differences could be accounted for either by the extent of mutation among clonal relatives or by usage of different V-region genes. Thus, functionally disparate anti-PPS antibodies can arise within individuals both by activation of independent clones and by intraclonal somatic mutation. For one pair of clonally related Fabs, the more extensively mutated V(H) was associated with lower affinity for PPS 14, a result suggesting that somatic mutation could lead to diminished protective efficacy. These findings indicate that the PPS repertoire in the adult derives from memory B-cell populations that have class switched and undergone extensive hypermutation.

Structural determinants of the human idiotype HibId-1.

The human antibody response to the capsular polysaccharide of Haemophilus influenzae type b is predominated by antibodies expressing a light-chain-associated idiotype designated HibId-1. HibId-1 is expressed by kappa light chains encoded by either the A2 or A18 variable region genes. In this report we use site-directed mutagenesis and molecular modeling to show that HibId-1 expression is determined by residues in the first and second complimentarity determining regions that are widely separated in the primary sequence, but closely juxtaposed by the tertiary folding of the mature light chain molecule. Of the known human light chains, only alleles of A2 and A18 encode these residues at these positions in their germline configuration. VIG10, a mouse monoclonal antibody of unknown specificity that expresses HibId-1, and 23F.2, an A2-utilizing Streptococcus pneumoniae 23F polysaccharide-specific human Fab fragment that lacks HibId-1, provide examples of the HibId-1 determinant both arising and being lost by somatic mutation. In addition, we show that the residues responsible for HibId-1 expression can be disassociated from those required for antigen binding.

Polysaccharide vaccines as probes of antibody repertoires in man.

Antibodies specific for capsular polysaccharide epitopes mediate immunity to encapsulated bacterial pathogens, and accordingly, vaccine development has focused upon the induction of these specificities. Efficacious vaccines, consisting of either polysaccharide alone or polysaccharide coupled to protein carriers, have been developed for a number of pathogens. Their clinical importance notwithstanding, these vaccines serve as model antigens to study the genetic and somatic forces molding adaptive immunity in man. In this article we review progress aimed at delineating the structure and dynamics of the human antibody repertoire to the Haemophilus influenzae type b polysaccharide (Hib PS), a system which has been studied from infancy to old age. Collectively, the data reveal a repertoire which is encoded by a relatively large number of germline variable (V) region gene segments, but which is typically expressed within individuals as a markedly restricted, oligoclonal population. One particular V domain has attained canonical status because of its high penetrance at the population level and its predominance in individual repertoires. Although this combining site is assembled in early infancy and retains its prominence throughout life, its frequency of expression, affinity and protective function are dictated by the molecular form of the Hib PS immunogen (vaccine). The determinants of Hib PS binding affinity can include both germline and somatically-acquired V region polymorphisms. We discuss how these properties of the Hib PS repertoire could impact immunity to Hib, and we consider the implications of these findings towards understanding the evolution of immunoglobulin germline V genes.

Polysaccharide binding potential of the human A2 or A18 kappa light chain homologues.

Antibodies having light (L) chains encoded by the kappaII-A2 variable region gene segment predominate in the human response to the Haemophilus influenzae type b polysaccharide (Hib PS). To determine whether the closely related homologue of the A2 gene, the kappaII-A18 gene, has the potential to contribute to the repertoire, we examined Hib PS binding to a series of recombinant Fab fragments having either A2 or A18 L chains isolated from a Hib PS-vaccinated adult. The ability to bind Hib PS resided exclusively with those Fab fragments having A2 and containing an insertional arginine at the variable-joining junction. Thus, despite the sequence similarity between A2 and A18, only A2 contributes to the canonical Hib PS paratope.

Role of kappa II-A2 light chain CDR-3 junctional residues in human antibody binding to the Haemophilus influenzae type b polysaccharide.

Abs using the kappaII-A2 V gene segment predominate the human Ab repertoire to the Haemophilus influenzae b (Hib) polysaccharide (PS). All A2 anti-Hib PS Abs sequenced to date possess a 10-amino acid L chain complementarity-determining region-3 (CDR-3) having an insertional arginine (Arg) at position 95a, the V-J junction. These findings suggest an essential requirement for this conserved Arg residue in determining Hib PS-binding affinity. We examined this requirement by performing chain recombination experiments in which a series of A2 L chains, differing at position 95a, were combined individually with an Fd region known to generate a Hib PS-combining site when paired with an A2-Arg(95a)-Jkappa1 V region. Hib PS binding of the recombinant Fabs was evaluated quantitatively using a radioantigen-binding assay. Fabs having A2 L chains with either Arg or lysine in position 95a in combination with Jkappa1 gave equivalent and strongest binding to Hib PS. Fabs having A2-Jkappa1 L chains with either tyrosine, glycine, alanine, leucine, serine, or threonine in position 95a, or having an A2-Arg(95a)-Jkappa3 L chain, gave intermediate binding. Fabs having A2-Jkappa1 L chains with glutamate or aspartate at 95a or with no junctional residue showed little or no Hib PS binding. These results demonstrate the importance of L chain junctional residue, as well as Jkappa usage and CDR-3 length, in determining Hib PS-binding affinity. Contrary to expectation, an Arg junctional residue is not essential for generating either high or intermediate affinity-binding sites.

Aging and the immune response to the Haemophilus influenzae type b capsular polysaccharide: retention of the dominant idiotype and antibody function in the elderly.

Anti-Haemophilus influenzae b polysaccharide (Hib PS) antibodies elicited in elderly subjects following conjugate vaccination expressed a light-chain variable-region (VL)-associated idiotype and had functional activities similar to those previously observed in children and younger adults. These findings indicate that advanced age is not accompanied by shifts in the major VL component of the Hib PS-specific repertoire or by diminution of the protective function of antibodies.

Human Fab fragments specific for the Haemophilus influenzae b polysaccharide isolated from a bacteriophage combinatorial library use variable region gene combinations and express an idiotype that mirrors in vivo expression.

To determine whether the human antibody (Ab) repertoire to the Haemophilus influenzae type b capsular polysaccharide (Hib PS) could be studied at the molecular level with phage display technology, we constructed a phage Fab library by using peripheral blood from a vaccinated adult. Phage were selected based on Hib PS binding. Two distinct Hib PS-specific phage clones were identified whose Fab fragments used the same V(H) region paired with two different V(L) regions. The V(L) regions were derived from two independent rearrangements of the A2c gene with Jkappa1, and both contained a nontemplated arginine codon at the V-Jkappa junction. The two A2 V gene segments differed from the A2c germ line sequence in 0 and 5 bases. The V(H) region consisted of the V(H)26 gene segment having 98% identity to the germline nucleotide sequence, a D region of 9 bases, and J(H)4b1. Usage of V(H)26 in combination with A2 V regions containing a junctional arginine is a predominant configuration of naturally occurring Hib PS-specific Abs. Liquid- and solid-phase assays showed that phage-derived Fab reacted with Hib PS and expressed HibId-1, an idiotype associated with the kappaII-A2 V region. These findings extend the database of V region polymorphisms that can contribute to the Hib PS repertoire and demonstrate that Hib PS-specific Fab fragments isolated from combinatorial phage libraries use V gene combinations which mirror the natural repertoire.

T lymphocyte proliferative responses induced by recombinant factor VIII in hemophilia A patients with inhibitors.

In this study we sought to determine whether factor VIII-reactive T lymphocytes were present in hemophilia A patients with inhibitor antibodies. Peripheral blood mononuclear cells (MNC) were obtained from 12 severe hemophilia A patients having high titer inhibitors, 4 severe hemophilia A patients without inhibitors and 5 normal male subjects. B cell-depleted MNC were cultured in serum-free medium in the absence or presence of 2 micrograms of recombinant human factor VIII (rFVIII) per ml, and cellular proliferation was assessed after 5 days of culture by measuring 3H-thymidine incorporation. rFVIII induced marked cellular proliferation in cultures of 4 of 12 inhibitor-positive hemophilia patients: fold increase over background (stimulation index, SI) of 7.8 to 23.3. The remaining 8 inhibitor-positive patients, the 4 hemophilia patients without inhibitors and the 5 normal subjects, all had lower proliferative responses to rFVIII, SI range = 1.6 to 6.0. As a group, the inhibitor-positive subjects had significantly higher proliferative responses to rFVIII than did the inhibitor-negative and normal subjects (p < 0.05 by t-test). Cell fractionation experiments showed that T lymphocytes were the rFVIII-responsive cell type, and that monocytes were required for T cell proliferation. Thus, rFVIII-reactive T lymphocytes are present in the peripheral circulation of some inhibitor-positive hemophilia A patients. These T cells may recognize FVIII in an antigen-specific manner and play a central role in the regulation of inhibitor antibody production.

Variable region sequences of a protective human monoclonal antibody specific for the Haemophilus influenzae type b capsular polysaccharide.

A hybridoma secreting a human immunoglobulin G2 kappa monoclonal antibody (MAb) specific for the capsular polysaccharide of Haemophilus influenzae type b (Hib) was isolated. This MAb, designated CA4, was bactericidal to Hib in vitro and protected infant rats from Hib bacteremia. Nucleotide sequence analysis of CA4 variable (V) region cDNA showed that the heavy (H)-chain V region was of subgroup III and was 96% identical to the VH germ line gene segment DP77 (V3-21). The light (L)-chain V region was of the kappa subgroup III and was 94% identical to the A27 (Humkv325) germ line gene, which is commonly used by rheumatoid factors and other autoantibodies. MAb CA4 did not have rheumatoid factor activity and did not react with histones, DNA, or chromatin. These findings identify an additional VHIII gene segment which can contribute to the anti-Hib capsular polysaccharide repertoire and demonstrate that a VL gene commonly encoding autoantibodies can be utilized for protective immunity.

Induction of a protective human polysaccharide-specific antibody response in hu-PBL SCID mice by idiotypic vaccination.

The human Ab repertoire to the Haemophilus influenzae type b (Hib) polysaccharide (PS) is dominated by Abs that use the kappa II-A2 VL region and that express an idiotype (Id) designated Hibld-1. In this study we determined whether a human Hib PS-specific Ab response could be induced by idiotypic manipulation. We prepared a bispecific vaccine consisting of the F(ab')2 fragment of a mAb specific for Hibld-1, coupled to the F(ab')2 fragment of a mAb specific for CD3, a component of the TCR complex. This bispecific idiotypic vaccine stimulated production of human Abs to Hib PS in severe combined immunodeficient mice engrafted with normal human adult PBLs. The induced Abs uniformly expressed Hibld-1 and protected neonatal rats from Hib bacteremia. Experiments using additional conjugates demonstrated that covalent coupling of the CD3-specific moiety to the anti-Id was required for immunogenicity in this model, a result suggesting that engagement of B cell Id and proximate delivery of T cell signals are both necessary for B cell activation and differentiation. These findings demonstrate that human Ids can serve as targets for induction of a protective anti-PS Ab response.

Interleukin 4-mediated induction of CD4+/CD8+ T cells during infancy.

Neonatal human CD4+ T cells will co-express CD8 on their surface following short-term culture with interleukin 4 (IL-4). Adult T cells do not respond in this manner. In this study we examine this phenomenon as a function of age and determine that IL-4 responsiveness decreases with time to approach adult levels at about 2 years. This phenomenon may be relevant to the documented ability of neonatal CD4+ T cells to function as suppressors.

Extracellular ATP stimulates interleukin-dependent cultured mast cells and eosinophils through calcium mobilization.

We examined the effect of ATP and related nucleotides on the changes in intracellular calcium ([Ca2+]i) in murine bone marrow-derived mast cells (BMMC) and human cord blood-derived eosinophils (EO) cultured in the presence of interleukins. ATP, ADP and AMP released a substantial amount of histamine and leukotriene C4 from BMMC, and EO showed locomotive activity in response to ATP, ADP and GTP. These reactions were accompanied with an increase in [Ca2+]i in BMMC and in EO. The rise in [Ca2+]i in BMMC induced by ATP or antigen at optimal concentrations was inclined to be persisting. On the other hand, these nucleotides induced a rapid and transient rise in [Ca2+]i in EO. Purified human peripheral EO also exhibited locomotive activity and an increase in [Ca2+]i in response to ATP. These results indicate that extracellular ATP activates interleukin-dependent cultured mast cells and EO through Ca2+ mobilization, and suggest that ATP, which is known to be released from activated platelets or autonomic nerves, may stimulate in vivo counterparts of these cultured inflammatory cells.

Changes in filament actin accompanying IgE-dependent and -independent histamine release from IL-3-dependent cultured human basophils.

When cord blood mononuclear cells were cultured in the presence of rhIL-3 for 5 weeks or more, 40-90% of cultured cells became morphologically mature basophils. We analyzed the kinetics of histamine release, changes in filament actin (F-actin), and movement of intracellular Ca2+ ([Ca2+]i) induced by IgE-dependent (anti-IgE) and -independent (fMLP) stimuli in these cultured basophils. Anti-IgE and fMLP released 24.5 +/- 5.4% and 14.5 +/- 4.5% histamine from the cells, respectively. Anti-IgE caused actin polymerization with a peak response at 15 min, which began much later than the elevation of [Ca2+]i. In contrast to anti-IgE stimulation, fMLP induced rapid actin polymerization with a peak response at 30 s in correlation with kinetics of histamine release. Our results indicate that cord blood-derived cultured basophils show similar cell functions to mature basophils, and are useful models with which to investigate the mechanisms of degranulation, specifically when a large amount of highly purified cells are required.

Interleukin 4 induces CD4+/CD8- to CD8+/CD4- transformation of human neonatal T cells by way of a double positive intermediate.

We have determined that IL-4 induces the generation of CD4+/CD8+ T cells in cultures of neonatal lymphocytes. Sorting, positive, and negative selection experiments indicate that these cells arise from a subpopulation of CD4+/CD8- cells present in the neonate but not in the adult. We have further determined that these IL-4 generated "double positive" cells further differentiate to express only the CD8 marker. Our findings suggest an undescribed and dramatic role for IL-4 in T cell differentiation.

Chemical mediators in hypersensitivity reactions.

The study of chemical mediators is essential in clarifying the mechanism of hypersensitivity reactions. Recent advances in this field have been made particularly with the arachidonic acid metabolites, several of which strongly contract smooth muscle, attract granulocytes to inflammatory foci and lead to increased vascular permeability. These functions play a crucial role in allergic reactions. Furthermore these mediators might work to modulate the intracellular network which mediates the complicated inflammatory process of hypersensitivity reactions.

Human cord blood lymphocytes do not simultaneously express CD4 and CD8 cell surface markers.

Several authors have reported finding significant numbers of immature T lymphocytes of the CD4/CD8 'double positive' phenotype in human cord blood. We have studied over 30 cord blood samples using directly conjugated reagents and have failed to detect such cells in any of the samples. Differences in staining methodologies that may account for this discrepancy are discussed.

Effects of human recombinant interleukin 5 and 3 on the differentiation of cord blood-derived eosinophils and basophils.

When mononuclear cells from umbilical-cord blood were cultured for 3 weeks, low concentrations of interleukin 3 supported the preferential growth of basophils, with eosinophils comprising a smaller proportion. These basophils contained 0.15-0.3 microgram histamine per 10(6) cells, and released histamine by the IgE-dependent and -independent stimuli. Interleukin 5 increased the number and proportion of eosinophils in a dose-dependent manner without affecting the proliferation of other cell types in the interleukin 3-supplemented cultures. These cultured eosinophils could be activated by platelet-activating factor.

A murine hybridoma with large cytoplasmic inclusions of kappa light chains.

A murine hybridoma cell line has been established that consistently forms large cytoplasmic inclusions. These structures bind antibody specific for mouse kappa L chain when stained in situ. SDS-PAGE analysis of isolated inclusion bodies produce a single protein band of approximately 26,000 Mr that reacts with anti-kappa antibody when transferred to nitrocellulose. No carbohydrate was detected in association with the purified protein. These data are consistent with the intracellular retention and deposition of complete kappa L chain protein.