Harry W. Schroeder

Structure and Function of Immunoglobulins

Immunoglobulins are heterodimeric proteins composed of 2 heavy and 2 light chains. They can be separated functionally into variable domains that bind antigens and constant domains that specify effector functions, such as activation of complement or binding to Fc receptors. The variable domains are created by means of a complex series of gene rearrangement events and can then be subjected to somatic hypermutation after exposure to antigen to allow affinity maturation. Each variable domain can be split into 3 regions of sequence variability termed the complementarity-determining regions (CDRs) and 4 regions of relatively constant sequence termed the framework regions. The 3 CDRs of the heavy chain are paired with the 3 CDRs of the light chain to form the antigen-binding site, as classically defined. The constant domains of the heavy chain can be switched to allow altered effector function while maintaining antigen specificity. There are 5 main classes of heavy chain constant domains. Each class defines the IgM, IgG, IgA, IgD, and IgE isotypes. IgG can be split into 4 subclasses, IgG1, IgG2, IgG3, and IgG4, each with its own biologic properties, and IgA can similarly be split into IgA1 and IgA2.

A Genomewide Screen in Multiplex Rheumatoid Arthritis Families Suggests Genetic Overlap with Other Autoimmune Diseases

Rheumatoid arthritis (RA) is an autoimmune/inflammatory disorder with a complex genetic component. We report the first major genomewide screen of multiplex families with RA gathered in the United States. The North American Rheumatoid Arthritis Consortium, using well-defined clinical criteria, has collected 257 families containing 301 affected sibling pairs with RA. A genome screen for allele sharing was performed, using 379 microsatellite markers. A nonparametric analysis using SIBPAL confirmed linkage of the HLA locus to RA (P < .00005), with lambdaHLA = 1.79. However, the analysis also revealed a number of non-HLA loci on chromosomes 1 (D1S235), 4 (D4S1647), 12 (D12S373), 16 (D16S403), and 17 (D17S1301), with evidence for linkage at a significance level of P<.005. Analysis of X-linked markers using the MLOD method from ASPEX also suggests linkage to the telomeric marker DXS6807. Stratifying the families into white or seropositive subgroups revealed some additional markers that showed improvement in significance over the full data set. Several of the regions that showed evidence for nominal significance (P < .05) in our data set had previously been implicated in RA (D16S516 and D17S1301) or in other diseases of an autoimmune nature, including systemic lupus erythematosus (D1S235), inflammatory bowel disease (D4S1647, D5S1462, and D16S516), multiple sclerosis (D12S1052), and ankylosing spondylitis (D16S516). Therefore, genes in the HLA complex play a major role in RA susceptibility, but several other regions also contribute significantly to overall genetic risk.

Motor Coordination via a Tug-of-War Mechanism Drives Bidirectional Vesicle Transport

The microtubule motors kinesin and dynein function collectively to drive vesicular transport. High-resolution tracking of vesicle motility in the cell indicates that transport is often bidirectional, characterized by frequent directional changes. However, the mechanisms coordinating the collective activities of oppositely oriented motors bound to the same cargo are not well understood. To examine motor coordination, we purified neuronal transport vesicles and analyzed their motility via automated particle tracking with nanometer resolution. The motility of purified vesicles reconstituted in vitro closely models the movement of LysoTracker-positive vesicles in primary neurons, where processive bidirectional motility is interrupted with frequent directional switches, diffusional movement, and pauses. Quantitative analysis indicates that vesicles copurify with a low number of stably bound motors: one to five dynein and one to four kinesin motors. These observations compare well to predictions from a stochastic tug-of-war model, where transport is driven by the force-dependent kinetics of teams of opposing motors in the absence of external regulation. Together, these observations indicate that vesicles move robustly with a small complement of tightly bound motors and suggest an efficient regulatory scheme for bidirectional motility where small changes in the number of engaged motors manifest in large changes in the motility of cargo.

High throughput sequencing reveals a complex pattern of dynamic interrelationships among human T cell subsets

Developing T cells face a series of cell fate choices in the thymus and in the periphery. The role of the individual T cell receptor (TCR) in determining decisions of cell fate remains unresolved. The stochastic/selection model postulates that the initial fate of the cell is independent of TCR specificity, with survival dependent on additional TCR/coreceptor “rescue” signals. The “instructive” model holds that cell fate is initiated by the interaction of the TCR with a cognate peptide-MHC complex. T cells are then segregated on the basis of TCR specificity with the aid of critical coreceptors and signal modulators [Chan S, Correia-Neves M, Benoist C, Mathis (1998) Immunol Rev 165: 195–207]. The former would predict a random representation of individual TCR across divergent T cell lineages whereas the latter would predict minimal overlap between divergent T cell subsets. To address this issue, we have used high-throughput sequencing to evaluate the TCR distribution among key T cell developmental and effector subsets from a single donor. We found numerous examples of individual subsets sharing identical TCR sequence, supporting a model of a stochastic process of cell fate determination coupled with dynamic patterns of clonal expansion of T cells bearing the same TCR sequence among both CD4+ and CD8+ populations.

Use and interpretation of diagnostic vaccination in primary immunodeficiency: a working group report of the Basic and Clinical Immunology Interest Section of the American Academy of Allergy, Asthma & Immunology.

A major diagnostic intervention in the consideration of many patients suspected to have primary immunodeficiency diseases (PIDDs) is the application and interpretation of vaccination. Specifically, the antibody response to antigenic challenge with vaccines can provide substantive insight into the status of human immune function. There are numerous vaccines that are commonly used in healthy individuals, as well as others that are available for specialized applications. Both can potentially be used to facilitate consideration of PIDD. However, the application of vaccines and interpretation of antibody responses in this context are complex. These rely on consideration of numerous existing specific studies, interpolation of data from healthy populations, current diagnostic guidelines, and expert subspecialist practice. This document represents an attempt of a working group of the American Academy of Allergy, Asthma & Immunology to provide further guidance and synthesis in this use of vaccination for diagnostic purposes in consideration of PIDD, as well as to identify key areas for further research.

Immunoglobulin VH clan and family identity predicts variable domain structure and may influence antigen binding.

Mammalian immunoglobulin VH families can be grouped into three distinct clans based upon sequence conservation in two of the three framework (FR) intervals. Through replacement/silent site substitution analysis, molecular modeling and mathematical evaluation of known immunoglobulin crystal structures, we demonstrate that this conservation reflects preservation of protein sequence and structure. Each clan contains a characteristic FR 1 interval that is solvent‐exposed and structurally separated from the antigen binding site. Families within a clan contain their own unique FR 3 interval that is capable of either influencing the conformation of the antigen binding site or interacting directly with antigen. Our results provide a structural context for theories that address differential use of VH families in the immune response.

Complement activation in factor D-deficient mice

To assess the contribution of the alternative pathway in complement activation and host defense and its possible role in the regulation of systemic energy balance in vivo, factor D-deficient mice were generated by gene targeting. The mutant mice have no apparent abnormality in development and their body weights are similar to those of factor D-sufficient littermates. Complement activation could not be initiated in the serum of deficient mice by the alternative pathway activators rabbit erythrocytes and zymosan. Surprisingly, injection of cobra venom factor (CVF) caused a profound and reproducible reduction in serum C3 levels, whereas, as expected, there was no C3 reduction in factor B-deficient mice treated similarly. Studies of C3 and factor B activation in vitro by CVF demonstrated that in factor D-deficient serum the α chain of C3 was cleaved gradually over a period of 60 min without detectable cleavage of factor B. CVF-dependent C3 cleavage in the deficient serum required the presence of Mg2+, whereas in normal mouse serum the presence of divalent cations was not required. These results suggest that in mouse proteolytic cleavage of factor B by factor D is not an absolute requirement for the zymogen to active enzyme conformational transition of CVF-bound factor B. Kinetics of opsonization of Streptococcus pneumoniae by C3 fragments was much slower in factor D-deficient serum, suggesting a significant contribution of the alternative pathway to antibacterial host defense early after infection.

Comparison of the efficacy of IGIV-C, 10% (caprylate/chromatography) and IGIV-SD, 10% as replacement therapy in primary immune deficiency ☆: A randomized double-blind trial

A novel method of large-scale chromatography has been developed to improve recovery and purity of immunoglobulin G (IgG) from pooled plasma. The current study compares safety, toxicity and efficacy of two intravenous immunoglobulin products: a novel formulation, IGIV caprylate/chromatography (IGIV-C; Gamunex, 10%) and a licensed solvent/detergent-treated product, Gamimune N, 10% (IGIV-SD). The study, a randomized, double-blind, parallel group, therapeutic equivalence trial, was conducted at 25 treatment centers in Canada and the United States. Patients (n=172) having confirmed chronic primary immunodeficiency (PID), aged 1-75 years, and receiving IGIV therapy were enrolled. For 9 months, patients were treated with IGIV-C or IGIV-SD in accordance with the patients individualized treatment regimen utilized before study entry. The primary endpoint was the proportion of patients with >or=1 validated acute sinopulmonary infection during the treatment period. Secondary endpoints included the proportion of patients with all infections, time to first infection, annual infection rates, lung function parameters, infusion-related safety and viral safety. The annual validated infection rate in the IGIV-C group was 0.18 compared to 0.43 in the IGIV-SD group (p=0.023). Nine patients receiving IGIV-C experienced validated infections, compared to 17 patients in IGIV-SD group (p=0.06). Acute sinusitis (validated plus clinically defined) was less frequent in the IGIV-C group (p=0.012). Presence of bronchiectasis did not affect efficacy. Adverse reactions were similar in frequency and severity in both groups. No evidence of viral transmission was observed. IGIV-C appears to be superior to IGIV-SD in preventing validated sinopulmonary infections, especially acute sinusitis, in patients with PID.

Development of the Expressed Ig CDR-H3 Repertoire Is Marked by Focusing of Constraints in Length, Amino Acid Use, and Charge That Are First Established in Early B Cell Progenitors

To gain insight into the mechanisms that regulate the development of the H chain CDR3 (CDR-H3), we used the scheme of Hardy to sort mouse bone marrow B lineage cells into progenitor, immature, and mature B cell fractions, and then performed sequence analysis on VH7183-containing Cμ transcripts. The essential architecture of the CDR-H3 repertoire observed in the mature B cell fraction F was already established in the early pre-B cell fraction C. These architectural features include VH gene segment use preference, DH family usage, JH rank order, predicted structures of the CDR-H3 base and loop, and the amino acid composition and average hydrophobicity of the CDR-H3 loop. With development, the repertoire was focused by eliminating outliers to what appears to be a preferred repertoire in terms of length, amino acid composition, and average hydrophobicity. Unlike humans, the average length of CDR-H3 increased during development. The majority of this increase came from enhanced preservation of JH sequence. This was associated with an increase in the prevalence of tyrosine. With an accompanying increase in glycine, a shift in hydrophobicity was observed in the CDR-H3 loop from near neutral in fraction C (−0.08 ± 0.03) to mild hydrophilic in fraction F (−0.17 ± 0.02). Fundamental constraints on the sequence and structure of CDR-H3 are thus established before surface IgM expression.

Forced usage of positively charged amino acids in immunoglobulin CDR-H3 impairs B cell development and antibody production

Tyrosine and glycine constitute 40% of complementarity determining region 3 of the immunoglobulin heavy chain (CDR-H3), the center of the classic antigen-binding site. To assess the role of DH RF1-encoded tyrosine and glycine in regulating CDR-H3 content and potentially influencing B cell function, we created mice limited to a single DH encoding asparagine, histidine, and arginines in RF1. Tyrosine and glycine content in CDR-H3 was halved. Bone marrow and spleen mature B cell and peritoneal cavity B-1 cell numbers were also halved, whereas marginal zone B cell numbers increased. Serum immunoglobulin G subclass levels and antibody titers to T-dependent and T-independent antigens all declined. Thus, violation of the conserved preference for tyrosine and glycine in DH RF1 alters CDR-H3 content and impairs B cell development and antibody production.