The HLA Complex
The human major histocompatibility complex (MHC), or human leukocyte antigen (HLA) complex, consists of many genetic loci, including seven loci that encode two distinct classes of highly polymorphic cell surface antigens that are co-expressed. These molecules bind and present processed peptide to circulating T-cell lymphocytes and are crucial to both cellular and humoral immune responses. The class I MHC molecules, HLA-A, HLA-B and HLA-Cw, and class II MHC molecules, DR, DQ and DP, are encoded in a ~3500 kbp segment of the short arm of chromosome 6p21.31 (Figure 1). Class I antigens are presented on all nucleated cells, where they act as cell surface heterodimers that primarily present peptides derived from the cytosol (viral and self peptides) to circulating CD8+ T cells. The class I cell surface heterodimer has one highly polymorphic alpha chain, with variable residues clustering within the peptide-binding cleft, which is encoded by exons 2 and 3 of the gene. The HLA class I molecules also act as ligands for killer immunoglobulin receptors (KIR), which regulate the cytotoxic activity of natural killer (NK) cells. HLA class II molecules are found on the surface of B cells, macrophages and other antigen presenting cells, where the alpha-beta heterodimer presents primarily exogenously derived peptides (bacteria and chemical toxins) to circulating CD4+ T cells. In class II molecules, the beta chain contains the highly polymorphic regions, which are localized to exon 2 of the gene and encode the peptide binding cleft.

Figure 1.  The Major histocompatibility complex (MHC) (from http://www.ebi.ac.uk/imgt/HLA/).

HLA & Hematopoietic Stem Cell Transplanatation
Matching class I and class II HLA antigens between recipient and donor reduces the incidence and severity of an alloreactive immune response when transplanting hematopoietic stem cells.  Over the last decade, molecular methods emerged as the most precise HLA typing approach, which many clinical laboratories now utilize. Retrospective and prospective hematopoietic stem cell transplantation (HSCT) studies have helped to refine the algorithms used to find the best allogeneic donor HLA match. The Trachtenberg Laboratory was integrally involved in the seminal National Marrow Donor Program (NMDP) examination of the role of HLA match in HSCT (Flomenberg et al., 2004). In this study, HLA-C was found to have a critical role  in engraftment , and HLA class I (A, B, Cw) and class II (DRB1, DQB1)  matching at allelic levels was associated with better outcome and survival in allogeneic transplants . Working with the CHRCO Blood and Marrow Transplanation Dept., the HLA & Immunogenetics Laboratory uses state-of-the-art molecular methods to characterize the HLA class I A, B, Cw and class II DRB1, DQB1 alleles at a resolution based on related or unrelated allogeneic donor status, according to an algorithm developed using current regulations from the gold-standard American Society of Histocompatibility and Immunogenentics (ASHI) and the National Marrow Donor Program (NMDP). The Laboratory also performs quantitative chimerism analysis of donor and recipient samples using peripheral blood, bone marrow or cell subsets (e.g. CD3, CD33), used to follow stem cell transplantation engraftment over time. The Trachtenberg Laboratory is also a leader in genotype analysis of killer immunoglobulin-like receptor (KIR) genes and the role of KIR in stem cell transplant outcome.

Umbilical cord blood (CB) has become an important alternative source of stem cells, as it presents diminished risk to the donor and diminished risk of graft-versus-host disease compared with marrow. Because of their immunologic immaturity, allogeneic umbilical CB stem cells may require less stringent HLA matching than BM or PB stem cells, which require quite rigorous HLA matching for HSCT.  Working with the CHORI Sibling Donor Cord Blood Project, the Trachtenberg Laboratory established optimum molecular HLA typing strategy and methods for determining class I HLA-A, -B and class II DRB1 antigens in the sibling umbilical cord blood samples. Our methods preserve as much as possible the already limited number of progenitor stem cells available from a single collection event for transplantation, and can identify gross maternal T cell contamination, both of which can be potential problems in CB transplantation.

HLA Heterogeneity in Disease Association Research
The HLA genes are the most polymorphic genes known in any mammalian system. The allelic diversity of the Class I and Class II loci is extensive, with many thousands of alleles described (for a current listing of alleles at all loci see http://www.anthonynolan.org.uk/HIG/). This extensive polymorphism allows for differential binding of peptide, and is therefore functionally significant in terms of disease susceptibility and progression. The high level of HLA polymorphism is maintained in populations by balancing selection, specifically pathogen-driven selection with heterozygote advantage. Populations tend to exhibit a distribution of frequencies of alleles and extended haplotypes particular to that group. This can confound HLA disease association studies that differ with respect to ethnic groups in cases and controls, making comparisons between studies more difficult. Concordant results between studies of different ethnic groups serves to support the HLA association for both groups, and discordant results may mean that the allele is simply a marker for the actual locus, or that the different ethnic groups have different HLA disease susceptibility alleles.

The MHC is an important host genetic risk factor in infectious and autoimmune disease, with associations to susceptibility or resistance in well over 50 different diseases, including many of viral origin. Immunologic susceptibility to viral infection in humans was originally discovered by Zinkernagel and Doherty, who determined that the HLA complex class I restriction with cytotoxic T-cell lymphocytes plays a major role in the immune response and destruction of virally infected cells. The HLA system has since been found associated with susceptibility or resistance to many different viruses and other infectious diseases, cancers, and autoimmune diseases. HLA is also known to play a role in reproductive health.  The Trachtenberg laboratory has projects examining the role of HLA in protection or susceptibility to HIV and disease progression to AIDS, infectious disease, cancer, autoimmune disease, pre-eclampsia and premature delivery.

HIV infection in susceptible hosts begins as a slow progressive degeneration of the immune system, characterized by a relentless decline of CD4+ T cells that as a rule eventually results in immunodeficiency, AIDS, and death. There are many clinical variations of HIV-1 disease and although several different host immunological factors have been implicated as important influences in the course of HIV transmission and disease progression, the HLA complex is one of the most significant.  Dr. Trachtenberg’s group used a population-based approach to examine the role of HLA in HIV disease progression. With colleagues Bette Korber (Los Alamos National Laboratory) and Steven Wolinsky (Northwestern University), Trachtenberg studied a large group of homosexual men enrolled in the Chicago component of the Multicenter AIDS cohort study (Trachtenberg et al., Nature Medicine, 2003). By examining the HLA “supertypes” (nine specific groups of HLA alleles defined by particular amino acids in pockets of the peptide binding groove) with levels of AIDS virus and CD4+ T cells over time, the group found that those patients with more common HLA supertypes progressed to AIDS more quickly than participants who had the rarer HLA supertypes. The virus was able to outwit the immune system in patients with the more common HLA supertypes, presumably because it had earlier encountered the more common HLA types in the population. The virus was, however, thwarted by the rarer HLA types. Those patients with more common HLA types tended to get higher overall viral loads more rapidly than those with the rarer HLA supertypes, indicating that HIV adapts to the most frequent HLA proteins in a population. This study thus provides direct evidence for frequency-dependent selection, giving a selective advantage for patients with rare HLA antigens.

The KIR Complex
The killer immunoglobulin-like receptors (KIR) are a family of inhibitory and activating MHC class I receptors expressed on natural killer (NK) cells and subpopulations of T cells (Figure 2 and Figure 3).  The inhibitory KIR, in partnership with alternative NK receptor complexes (CD94:NKG2A), ensure that NK cells are tolerant of healthy autologous cells and responsive to cells with compromised MHC class I expression, as occurs frequently in virus-infected and tumor cells.  Although their ligands and functions are less clearly defined, the activating KIR are hypothesized to contribute to the activation of NK cells in response to infection and malignancy.  

Figure 2.  KIR Molecules are polygenic, polymorphic and reside on NK and some T cells. The KIR molecules interact with polymorphic HLA class I molecules. (Figure by Kathleen Houtchens).

The KIR gene family is part of the leukocyte receptor complex (LRC), located on human chromosome 19q13.4 (Figure 4). The KIR complex comprises a tandem array of highly homologous genes, which exhibits haplotypic variation in gene content as well as polymorphism of the individual KIR genes. As a consequence of these variations, unrelated individuals usually differ in KIR genotype.  KIR, either alone or in combination with HLA class I, have been correlated with a variety of diseases and syndromes. These include the outcome of bone marrow transplantation, the progress and severity of virally transmitted disease, the probability of pre-eclampsia during pregnancy, and susceptibility to autoimmune diseases such as psoriatic arthritis, scleroderma, sarcoidosis and type 1 diabetes. Collectively, these studies make a convincing case that interactions between variable HLA class I ligands and variable KIR have considerable impact on human health. Consequently, a better understanding of the underlying mechanisms could influence future clinical practice. The Trachtenberg Laboratory is currently examining the role of KIR and HLA ligands in HIV infection and disease progression to AIDS, stem cell transplantation and malignancy, autoimmune disease, pre-eclampsia and premature delivery.

Figure 3.  KIR Structure. KIR contain two or three extra-cellular immunoglobulin-like domains and have inhibitory or stimulatory properties depending on the isoform of the intracellular tail.
(Figure by Steven Marsh, Anthony Nolan Institute, UK).

Figure 4.  KIR Gene Complex and Haplotype. The KIR gene complex is located on human chromosome 19q13.4. The KIR haplotype A is the simplest KIR haplotype with one stimulatory gene (KIR2DS4), although in rare cases no stimulatory genes are found on this type. The KIR haplotype B is generally characterized by more than one stimulatory gene. (Figure by Steven Marsh, Anthony Nolan Institute, UK).

 Small cohorts and the low-resolution genotyping of the KIR complex, which has mainly involved determination of the presence and absence of KIR genes, has limited interpretation of the published studies on clinical associations and population comparisons of KIR. The key problems are that the established methods of KIR genotyping are labor intensive and require more DNA (300 ng – 5 ug) than is compatible with many of the samples that are available and informative to study.  A new method that combined speed, economical consumption of DNA and a resolution that distinguishes all KIR alleles was needed. To this end, the Trachtenberg group developed a novel assay to genotype the KIRcomplex using Matrix Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) mass spectrometry (MS) (Houtchens et al., 2007). The technique can accurately and rapidly detect single nucleotide polymorphisms (SNPs), the dominant feature of KIR variation. The method is applicable to high-resolution and high-throughput KIR genotyping, utilizes minimal amounts of DNA (40-100 ng), and is capable of discovering novel alleles, all of import to epidemiological studies and those using rarer archived specimens.