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The field of immunogenetics explores how genetic variations influence immune responses and susceptibility to disease. In a recent SEQanswers webinar, Oscar Rodriguez, Ph.D., Postdoctoral Researcher at the University of Louisville, and Ruben Martínez Barricarte, Ph.D., Assistant Professor of Medicine at Vanderbilt University, shared recent advancements in immunogenetics. This article discusses their research on genetic variation in antibody loci, antibody production processes, and newly identified immunodeficiency mechanisms.
V(D)J Recombination in Antibody Diversity
Rodriguez began his presentation by reviewing antibody generation. Antibodies are essential to the immune system’s ability to recognize and neutralize pathogens. They are made up of heavy and light chains, with a variable region that binds to pathogens and a constant region that activates immune cells. Genetic recombination of variable (V), diversity (D), and joining (J) genes in the IGH (chromosome 14), IGK (chromosome 2), and IGL (chromosome 22) loci facilitates the production of a diverse set of antibodies.
Rodriguez highlighted that significant genetic variations, such as copy number variations and structural polymorphisms, exist within these loci. For example, a variant of IGHV1-69 can protect against influenza in mice, while another variant affects response to an HIV vaccine. However, the complexity of the IGH locus, with its repeats and duplications, has made it challenging to study using traditional short-read sequencing. To overcome this, Rodriguez’s team developed a method using long-read sequencing from PacBio, enabling high-accuracy, haplotype-specific assemblies and the detection of single nucleotide polymorphisms (SNPs) and structural variants.
In a recent study, Rodriguez applied this long-read approach to sequence the IGH locus in 154 individuals, identifying substantial structural variations. Rodriguez emphasized, “We detected more than 20,000 single nucleotide polymorphisms, of which 8,000 were common. And of these common SNPs, 19% were missing from dbSNP.” While analyzing the antibody repertoire of these individuals, the team measured gene usage—the frequency of different genes selected during VDJ recombination. They observed significant variability between individuals in gene selection frequency, finding that certain SNPs correlated with differences in gene usage, suggesting that these genetic variations directly shape antibody production.
Additionally, Rodriguez’s team investigated whether these variations impacted regulatory mechanisms, such as transcription factor binding sites linked to chromatin structure. They observed an enrichment of genetic variants in regions involved in chromatin organization. This suggested that some observed antibody diversity may result from changes in 3D genome structure, which influences gene accessibility during recombination.
B-Cell Development and Its Influence on the Antibody Repertoire
Another focus of the talk was the influence of B-cell development on the antibody repertoire. During B-cell maturation in the bone marrow, antibodies are tested for proper function and non-reactivity to self. Although receptor editing can eliminate self-reactive antibodies, recent data suggest that genetic factors may consistently play a more significant role in determining the heavy chain repertoire than developmental processes.
To explore this further, the group sequenced antibody repertoires from developing B cells. They found that light chains changed more significantly during development than heavy chains. “We observed minimal changes to the heavy chain antibody repertoire across B-cell development,” Rodriguez noted. This finding suggests that genetic variations in the IGH locus may play a larger role in shaping the final heavy chain antibody repertoire than previously thought. Further testing revealed that certain IGH genetic variants influence the selection of light chain genes as well, likely due to the need for compatible heavy-light chain pairing. In some cases, SNPs in the IGH locus were linked to the selection of specific light chain genes, demonstrating a potential trans effect of IGH variants on overall antibody repertoire diversity.
Overall, Rodriguez’s work demonstrated that genetic variations in the IGH locus strongly impact the antibody repertoire, influencing the selection of both heavy and light chains. These genetic influences appear to have a more significant effect than developmental changes during B-cell development. “Now, the lab is beginning to apply this framework to antibody- or adaptive-mediated diseases such as autoimmune disorders and vaccine responses,” Rodriguez concluded.
Investigating Inborn Errors of Immunity
Barricarte shared recent findings from his research on inborn errors of immunity (IEI), a group of genetic disorders that compromise immune function. These monogenic diseases predispose individuals to infections, autoimmunity, and cancer. Through collaborations with an international network of clinicians, Barricarte’s team recruits patients with IEI and performs various sequencing techniques and multi-omics approaches to identify mutations that lead to immune dysfunction.
“This work is important because it allows for the genetic diagnosis of patients, genetic counseling of families, and the application of preventive and personalized medicine for individuals at risk,” Barricarte stated. “In addition, these patients give us a key to understand human immunology without relying on artificial models.”
Genetic Mutation and Immune Dysfunction
In this study, Barricarte’s lab investigated a unique cohort of seven patients from six countries who shared a rare form of combined immunodeficiency characterized by recurring severe infections. All seven had a T95R mutation in the transcription factor IRF4 gene. This mutation was unique because it was not inherited in most cases, indicating it was a de novo mutation. To fully understand its impact, the team investigated both the immunological and molecular disruptions it causes.
Starting with the immunological effects, Barricarte explained how his team performed cytometry by time-of-flight (CyTOF) analysis, revealing that the patients lacked memory B cells and plasmablasts. Without memory B cells, these individuals couldn’t effectively produce antibodies, explaining their susceptibility to infections. Single-cell RNA sequencing showed that the B cells in patients followed an altered developmental pathway, affecting their ability to produce antibodies. Although T cells in these patients showed normal proliferation, they had a reduced ability to secrete essential proinflammatory cytokines, such as IL-2 and TNF. The lack of antibody production, combined with cytokine impairment, formed a clear immunological profile consistent with the patients’ symptoms.
The team then explored how this mutation affects IRF4 function. Structural analysis and 3D modeling showed that the mutation, located in IRF4’s DNA-binding domain, increased the protein’s affinity for DNA. The mutated protein bound DNA more tightly than the wild type, as demonstrated by techniques like EMSA and single-molecule microscopy.
However, the T95R mutation exhibited unusual “multimorphic” behavior, combining gain-of-function, loss-of-function, and neomorphic effects. While the mutated IRF4 bound standard DNA motifs with greater affinity, it also gained the ability to bind novel GATA domains, which are not typically targeted by wild-type IRF4. Despite binding DNA more effectively, the mutant did not always activate gene transcription in the same way, indicating a complex functional alteration.
To further validate these findings, Barricarte’s team performed chromatin immunoprecipitation sequencing (ChIP-seq) and RNA sequencing on patient cells. They found distinct binding patterns in mutant versus wild-type IRF4, with some genes showing increased expression only in the presence of the mutation. For example, the chemokine gene CXCL13, which promotes immune cell recruitment, was upregulated in patients, confirmed by high plasma levels detected through ELISA. Barricarte explained, “This combination of gain-of-function, loss-of-function, and new functions defines a new mechanism of human disease.”
The findings led the team to coin a new term, “Multimorphic IRF4 Combined Immunodeficiency” (MICI), for this condition. MICI demonstrates a complex interplay of hypermorphic, hypomorphic, and neomorphic functions within a single mutation, significantly impacting immune cell differentiation and cytokine production. The team’s ongoing work includes developing two mouse models replicating the patient phenotype to further understand these types of immunodeficiencies.
Looking Ahead in Immunogenetics
The insights presented by Rodriguez and Barricarte demonstrate how advanced sequencing techniques can be applied to study genetic variation and mutations that shape the immune system and its ability to defend against disease. Their full presentations are available for on-demand viewing. Watch here.