Dan Geraghty

Geraghty Lab Genetics summary

Knowledge of DNA sequence variation promises to help us understand how genetic variability gives rise to functional variability and in so doing revolutionize the development of strategies to combat and prevent disease. Single nucleotide polymorphisms (SNPs) are stable, inherited, biallelic, single base pair differences which are present in the human genome at a density of 1 to 10 per 1000 nucleotides. It is anticipated that SNPs will account for much of the functional heterogeneity in gene expression and protein activity exhibited in the human population. Susceptibility to or protection from a number of diseases, particularly those of autoimmune etiology, has been associated with specific alleles of the human major histocompatibility complex (MHC) otherwise known as the HLA complex. Interestingly, the precise molecular defects in the HLA genes that are associated with disease are unknown, and the notion that non-HLA genes, within the same chromosomal region, are involved remains a significant possibility. Furthermore, the participation of immune regulatory loci resident at other genomic sites has been little studied at the genetic level. With the advent of modern genomics technology, the opportunity is now available to study variation in the MHC as a whole and, simultaneously, combine this with the genetics of a plethora of other genes contributing to the immune response.

The Geraghty lab is currently examining the relationship between the genetics of the immune response and human health using a simple three-step paradigm. The first step is to identify genetic variation within relevant genomic regions using state of the art genomic sequencing technology. This data is then taken forward into the second step and used to direct the development and application of high throughput methodologies for detection of the identified variation. Once robust methods are established, sufficiently large numbers of individuals can be examined to correlate variation with phenotype (e.g. disease susceptibility or resistance) for a variety of disorders to constitute the third step.

Building on our prior work determining the human MHC genomic sequence, we have identified over a thousand SNPs spanning the MHC and have begun to develop assays that are amenable to high-throughput detection. To expand the potential for study, we have also determined the genomic sequence of the Killer cell Ig-like receptor (KIR) locus. The cell surface expressed KIR gene products interact directly with HLA class I molecules to regulate the activity of natural killer cells and to a smaller degree, T cells. Analogous to HLA class I and II, the KIR genes have significant allelic polymorphism, although unlike HLA almost no functional examination of these polymorphisms has been carried out. We have been deciphering haplotypic variation at KIR in order to establish a primary level of variation. These data are being combined with second level allelic variation to establish detection methodologies amenable to the unique characteristics of this gene family.

We are initiating a new project to collect polymorphism data from a large number of additional immune regulatory loci by defining high-density SNP maps from over 100 individual loci and constructing associated databases. By combining an immune-specific focus supplemented with existing genome-wide efforts at identifying SNPs we expect to assemble virtually complete datasets of relevant allelic variation. This effort is being coordinated with the establishment of a Genetics Reference Laboratory that will be sanctioned with the service of genomic sequencing and high-throughput genotyping. This lab and the correlative population data it can derive will in turn facilitate our overall plans to directly link geneticsto human health.

Given the ability to access genetic data from large numbers of individuals, the third step of coordinating genetic data with phenotype of course requires access to appropriate populations of patients and controls. Therefore we are working in collaboration with an international group of scientists under the auspices of the International Histocompatibility Working Group (www.IHWG.org). A unique advantage of the international collaboration afforded by the IHWG is the broad access it provides to biological materials and skilled investigators. The foundation of the group is an NIH-funded program consisting of 11 research projects and 5 core components which share technology and resources for data collection and analysis. Through collaboration both within the program and with a much larger international network of investigators a variety of scientific issues are being investigated, including studies of several HLA-related diseases. We believe that by combining immune-related genetic data and technology with patient samples and resources from the IHWG, powerful new insights into the genetics of medically important problems will be forthcoming.


Geraghty Lab Immunobiology summary

Pregnancy represents one of the most interesting examples of immune accommodation seen in mammalian biology. The placenta provides a barrier between the mother and child, preventing maternal immune cells from entering the baby’s compartment at a time when its own immune system is immature or absent. Trophoblast cells of the placenta invade deep into the maternal uterine tissue to establish a life giving connection with the maternal blood supply. Human placentation is critically dependent on the ability of the trophoblast to evade recognition and elimination by maternal T cells and NK cells, both of which are present in the early pregnancy decidua. The mechanisms which operate to protect the placental cells from maternal immune attack, are interesting in their own right, but also may provide important information about specific reproductive diseases, and contribute towards a better understanding of immune tolerance. Thus, a comprehensive study of this subject is vital.
The Geraghty lab has been studying the biochemistry and immunology of the HLA-E, F, G molecules with a special focus on how they act individually, and interactively, to help accommodate the allogeneic child to the maternal immune response. Studies of protein expression and peptide binding have suggested both unique and interacting functional roles for HLA-E, F, and G in manipulating a maternal immune response. All three molecules are expressed in characteristic patterns on overlapping subsets of placental cells, some of which are in direct contact with maternal tissue and immune cells. HLA-E was shown to interact with CD94/NKG2 lectin-type NK receptors exerting either an activatory or inhibitory reaction depending on which of the related NKG2 molecules has combined with CD94. Further, HLA-E, which must bind peptide derived from other HLA class I in order to function, gains a novel function when it binds the unique peptide that only HLA-G can provide. This novel function is very suggestive of the unique role that HLA-E might be playing in the placenta. HLA-G in turn may interact exclusivelywith a unique receptor on NK cells thereby influencing the activity of these cells when they are present in the maternal decidua. HLA-F appears to be expressed most interestingly in the maternal placenta where it may appear on the cell surface of only those placental cells that are in direct contact with the maternal decidua. Peptide binding studies of these molecules expressed in vivo provide further indirect evidence that each is molded for a unique role to modify the maternal immune response.
In focused biochemical studies of HLA-E allelic polymorphism, we have uncovered functional differences that can manifest themselves in a peptide-dependent manner. Thus one of two HLA-E alleles is expressed at higher levels at the cell surface than its counterpart, with surface levels correlating with functional activity. Biochemical studies show that the reason for these differences is not apparent from the crystal structure, but is related to the differing stability of each heavy chain-peptide complex. Further, each allele shows differing affinities with distinct peptides in accord with the differing stability and expression levels. These observations may help to explain the hypothesized balancing selection that may be acting to maintain both alleles at nearlyequal frequencies.
Our current working hypotheses concerning HLA-E, F, G in the accommodation of the maternal immune response can be summarized as follows:

The HLA-E/G nonamer complex interacts with a unique receptor. This interaction stimulates NK to secrete cytokines of specific benefit for the implanted embryo. This response may be coordinated with a general NK inhibitory role by interaction with CD94/NKG2A.

HLA-G interacts with an inhibitory and a noninhibitory KIR receptor to achieve a balance to the activation signal conferred by HLA-E/G nonamer, resulting in a net inhibition of lysis while allowing other activatory pathways to proceed (e. g. cytokine secretion). HLA-G is unlikely to participate in antigen presentation for T cell immunity in the placenta.

HLA-F may act as a nonpolymorphic class I in a role originally envisioned for HLA-G, that being to present foreign antigen from placental cells to the maternal immune system. Alternatively and perhaps more likely, a role in the regulation of immune response through interaction with novel immune receptors either overlapping with, distinct from, or both, to those interactions of the HLA-E and HLA-G molecules.

Some of the active studies derivative of these hypotheses include:

1) Determining the factors that control HLA-F expression at the cell surface,
2) ligand interaction of HLA-F,
3) possible function of HLA-F as an intracellular agent,
4) the expression of all three molecules on tumor lines and tissues as a possible means of accommodating the immune response,
5) further demonstrating the functional differences of HLA-E alleles and their consequence in disease or marrow transplant outcome.