HLA-A

Overview
HLA-A a human leukocyte antigen belongs to the MHC class I heavy chain receptors. The HLA-A is a heterodimeric receptor consisting of an HLA-A mature gene product and β2-microglobulin. The mature A chain is anchored in the membrane. MHC Class I molecules, such as HLA-A, are expressed in nearly all cells, and present small peptides to the immune system which surveys for non-self peptides. As in most mammalian populations, MHC Class I molecules are extremely variable in their primary structure, and HLA-A is ranked among the genes in humans with the fastest evolving coding sequence. After typing millions of individuals, as of 10/15/2007 617 variant alleles have been identified, encoding for 486 protein isoforms.

Function
MHC Class I molecules present smaller peptides, generally 9mers but longer molecules are tolerated, to the immune system. Several target cells include CD8 + T-lymphocytes. In response to signalling these lymphocytes result in apototic cell death. This mechanism is the result of responses to viral infection or intracellular microbial infections in which, as a means of preventing propagation, affected cells are killed and the antigens are presented to the immune system for Class II presentation and antibody development. Over a short period of time antibodies develop that can neutralize the ability of viruses and invasive bacteria to invade cells.

Structure and Serology
The HLA-A chain forms a binding cleft much like the MHC Class II molecules, the sides of the cleft are composed of alpha helices, the base is beta sheet and one end the relative closure limits the optimal length of peptide.

To the right is a table of serotypes of HLA-A and there general relationships.

Nomenclature
HLA alleles and specificity. Some Allele groups have been updated with recent information from the IMGT/HLA Database

Explanation - within each allele group there are alleles that are recognized by the serological typing for that group (e.g. A24-serotype) some within the group may also recognize the broad antigen typing (A9, A10, A19, A28) or only the broad antigen typing, some by alternative serological within the group (e.g. A2403), and some by no serological method. Obviously some groups are more closely related than other groups, and this is often reflected in broad antigen reactivity.

Diseases by Haplotype
A*02:Cw*16 : higher viral load in HIV A*23:B*14 : higher viral load in HIV A*23:Cw*07 : higher viral load in HIV A*30:Cw*03 : higher viral load in HIV

Historical Guide to Understanding Nomenclature
The naming of HLA "antigens" is deeply rooted in the discovery history of their serotypes and alleles. There is no doubt that, except to an experienced HLA geneticist or immunologist, HLA terminology is bewildering

Perspective is important to an understanding this system.

Clinically, the point was to explain illness within the patients, who were transplant recipients. From this perspective, the cause of rejections are antigens, in the same way bacterial antigens can cause inflammatory response.


 * Lymphoid "antigens" became an experimental artifact of medical techniques (i.e., of transplantation). More simply, a familiarity with the human immune system resulted understinding that, in allograft (different graft) rejection, the cause was antibody production to allotypic proteins in donor tissue.


 * HLA gene products (ie, antigen-presenting, cell-surface receptors) do not appear to have evolved to be transplantation antigens, nor to interfere with transplantation, organ transplantation being unknown until 1960. The HLA genes are much older. Variation of HLA has led to an estimate that they are at least 60 million years in age for humans (DRB1)

The scientific problem has been to explain the natural function of a molecule, such as a self cell-surface receptor involved in immunity. It also seeks to explain how variation developed (perhaps by evolutionary pressure), and how the genetic mechanisms works (dominant, codominant, semidominant, or recessive; purifying selection or balancing selection).

Transplantation and transplant rejection
In the early 1960s, some physicians began more aggressive attempts at organ transplantation. Knowing little about compatibility factors, they attempted transplantation between humans and even between non-humans and humans. Immunosuppressive drugs worked for a time, but transplanted organs would either always fail or the patients would die from infections. Patients received skin, white blood cell or kidney donations from other donors (called allografts, meaning 'of different genetics' graft). If these allografts were rejected, it was found that the 'rejection' response was accompanied by an antibody mediated agglutination of red blood cells (See figure). The search for these cell surface antigens began. The process by which antibodies reduced function several fold.
 * Acute rejection - Antibodies could attract lymphocytes and cause them to lyse cells via the immune system's classical complement pathway
 * Antibodies could bind to and alter function (eg, flow of a fluid, or prevention of binding of ligands to receptors)
 * Cytokine responses that cause systemic responses.

Different antigens can be identified
In the accompanying figure, two similar haplotypes (unknown to early clinicians) are identical, except for the one antigen in the top haplotype. The transplant may not be rejected, but if rejection does occur that antigen in the donor tissue may have induced the dominant alloreactive antibody in the recipient.

Assaying Antiserum
Hemagglutination assay. In generating an immune response to an antigen, the B-cells go through a process of maturation, from surface IgM production, to serum IgM production, to maturation into a plasma cell producing IgG. Graft recipients who generate an immune response have both IgM and IgG. The IgM can be used directly in hemagglutination assays, depicted on the left. IgM has 10 antigen binding regions per molecule, allowing crosslinking of cells. An antiserum specific for HLA-A3 will then agglutinate HLA-A3 bearing red blood cells if the concentration of IgM in the antiserum is sufficiently high. Alternatively, a second antibody to the invariable (Fc) region of the IgG can be used to crosslink antibodies on different cells, causing agglutination.

Complement fixation assay. The complement fixation test was modified to assay Antiserum mediated RBC lysis.

Chromium release assay. This assay measures the release of (biological) radioactive chromium from cells as a result of killer cell activity. These cells are attracted to class I antigens that either carry foreign antigens, or are foreign to the immune system.

The role of haplotypes in identifying antigens
Each person has two HLA haplotypes genes, one from each parent. The haplotype frequencies in Europeans are in strong linkage disequilibrium. This means there are much higher frequencies of certain haplotypes relative to the expectation based on serotype (or allele) frequencies. This aided the discovery of HLA antigens, but was unknown to the pioneering researchers,.

In the table, on the right, a fortuitous transplant between two unrelated individual has resulted in an antiserum alloreactive to a single antigen. This allows researchers to match at least one antigen. Donors with A3 can be distinguished from recipients that lack A3.

In the case of the 5th example, there are several combinations, for example A2-Cw7-B7/A1-Cw7-B8, A2-Cw7-B7/A2-, A10-Cw7-B8. Given the distribution of haplotype in European Americans it is possible to estimate the probability of a random appearance of a single allotypic antigen. The most readily detected antigens are A3, A2, A1, A9, A10, and A11. Thus, the order of the antigens detected is largely a function of haplotype frequencies that could be combined to expose single antigen specificity when the highest probability is multiple specificities. Very rare halotype alleles in this population tend to have been identified much later, in other populations.

In the next stage researchers are capable of matching 3 alleles (unknown as the HLA-A) but not the B except through linkage with A. Occasionally A recombined with another B and resulted in a B allele mismatch.

In this instance, the A1/A2, A2/A3, A1/A3 are matched, decreasing the probability of a rejection because many are linked to a given haplotype. Occasionally the 'recombinant' A2-Cw7-B8 will cause alloreactivity to B8 if it was in the donor, or B7 if in the recipient.

This linkage disequilibrium in Europeans explains why A1, A2, A3, "A7"[B7], and "A8"[B8] were identified first. It would have taken substantially longer to identify other alleles because frequencies were lower, and haplotypes that migrated into the European population had undergone equilibration or were from multiple sources.

This is the genetic background against which scientists tried to uncover and understand the histocompatibility antigens.

A list of antigens created
In the late 1960's, scientist began reacting sera from patients with rejecting transplants to donor or 'third party' tissues. Their sera (the liquid part of the blood when blood clots) was sensitized to the cells from donors - it was alloreactive. Serum is rich in antibodies and can react to specific, inoculated antigens, becoming an antiserum. An alloreactive antiserum could have strong reaction with the cells from one person (e.g., the transplant donor), mild reaction to another's cells, and no reaction to a third's cells (e.g., a close relative). Likewise, a different alloreactive antiserum might not react with the first, show moderate reaction to a second, and strong reaction to the third person's cells.

As a result of this complex reactivity, scientists were able to identify 15 antigens. These were assigned, a simple number, from 1 to 15. At first these 15 antigens were called the Hu-1 antigens and tentatively tagged as gene products of the Human equivalent of the mouse histocompatibility locus. In 1968, it was discovered that matching these antigens between kidney donor and recipient improved the likelihood of kidney survival in the recipient. The antigen list still exists, although it has been reorganized to fit what we have since learned about genetics, refined, and greatly expanded.

Lymphocyte bearing antigens recognized
As the study of these 'rejection' sera and "allo"-antigens progressed, certain patterns in the antibody recognition were recognized. The first major observation, in 1969, was that an allotypic antibodies to "4" ("Four") was only found on lymphocytes, while most of the antigens, termed "LA", recognized most cells in the body.

This group "4" antigen on lymphocytes would expand into "4a", "4b" and so on, becoming the "D" series (HLA-D (Class II) antigens) DP, DQ, and DR. This is an interesting history in itself.

The Hu-1 antigens were renamed the Human-lymphoid (HL) alloantigens (HL-As). Alloantigen comes from the observation that a tolerated protein in the donor becomes antigenic in the recipient. This can be compared with an autoantigen, in which a person develops antibodies to one or more of their own proteins. This also suggested the donor and recipient have a different genetic makeup for these antigens. The "LA" group thereafter was composed of HL-A1, A2, A3, A5, A6, A7, A8, A9, A10, A11, A12, A13, A14 and A15 until further divisions and renaming were necessary. Some of the antigens above, for example HL-A1, are similar to HLA-A1, as they are the same serotype. Some of the above, like A5, are not mentioned within the last few years, as they have been renamed.

Subclassification of lymphoid antigens
A series of tests on cultured cells revealed that, within the "LA" group, a donor tissue might have some antigens but not others. For example, an antiserum may react with patterns (on a given tissue):


 * A1, A2, A7, A12
 * A1, A3, A7, A8
 * A1, A11, A8, A5
 * A1, A8
 * A2, A3, A7, A12
 * A2, A11, A
 * A2, A7, A12
 * A3, A11, A7, B5
 * A3, A7
 * A11, A5

But fail to react in the following patterns:


 * A1, A2, A3, ...
 * A1, A2, A11
 * A2, A3, A11
 * . . . A7, A8, A12

Series "A"
If 2 members of the series (A1, 2, 3, 9, 10, 11) were typed, a reaction with a third member of the series to the donor was not observed. This 'exclusivity' identified series "A". One might notice the simarities of this numeric series with the HLA-A series, as series "A" antigens are the first six members of HLA-A. Inadvertently, the scientist had discovered an antibody set that recognized only gene products from one locus, the "antigens" being the gene products. The implication is that an alloreactive antisera can be a tool for genetic identification.

Series "B"
Not long after the series A antigens were separated from the (rapidly expanding) list of antigens, it was determined another group also could be separated along the same logical lines. This group included HL-A5, A7, A8, A12. This became the series "B". Note the similarity of Series "B" to the first few members HLA-B serotypes. The names of these antigens were necessarily changed to fit the new putative series they were assigned to. From HL-A# to HLA-B#. The problem was that the literature was using "A7" and would soon be using "B7" as short hand for HLA-B7.

Pseudo-series "w"
Since it was now certain, by the early 1970s, that the "antigens" were encoded by different series, implicit loci, numeric lists became somewhat cumbersome. Many groups were discovering antigens. In these instances an antigen was assigned a temporary name, like "RoMa2" and after discussion, the next open numeric slot could be assigned, but not to an "A" or "B" series until proper testing had been done. To work around this problem a 'workshop' number "w#" was often assigned while testing continued to determined which series the antigen belonged to.

Series "C"
Before too long, a series "C" was uncovered. Series C has proved difficult to serotype, and the alleles in the series still carry the "w" tag signifying that status; in addition, it reminds us that Series C were not assigned names the same way as Series A and B, it has its own numeric list Cw1, Cw2, Cw3.

Serotype group expansion and refinement
By the mid 1970s, genetic research was finally beginning to make sense of the simple list of antigens, a new series "C" had been discovered and, in turn genetic research had determined the order of HLA-A, C, B and D encoding loci on the human 6p. With new series came new antigens; Cw1 and 2 were quickly populated, although Cw typing lagged. Almost half of the antigens could not be resolved by serotyping in the early 90's. Currently genetics defines 18 groups.

At this point, Dw was still being used to identify DR, DQ, and DP antigens. The ability to identify new antigens far exceeded the ability to characterize those new antigens.

As technology for transplanation was deployed around the world, it became clear that these antigens were far from a complete set, and in fact hardly useful in some areas of the world (eg, Africa, or those descended from Africans). Some serotyping antibodies proved to be poor, with broad specificities, and new serotypes were found that identified a smaller set of antigens more precisely. These broad antigen groups, like A9 and B5, were subdivided into "split" antigen groups, A23 & A24 and B51 & B52, respectively. As the HL-A serotyping developed, so did identification of new antigens.

Genetic identification
In the early 1980's, it was discovered that a restriction fragment segregates with individuals who bear the HLA-B8 serotype. By 1990, it was discovered that a single amino acid sequence difference between HLA-B44 (B*4401 versus B*4402) could result in allograft rejection. This revelation appeared to make serotyping based matching strategies problematic if many such differences existed. In the case of B44, the antigen had already been split from the B12 broad antigen group. In 1983, the cDNA sequences of HLA-A3 and Cw3 All three sequences compared well with mouse MHC class I antigens. The Western European HLA-B7 antigen had been sequenced (although the first sequence had errors and was replaced). In short order, many HLA class I alleles were sequenced including 2 Cw1 alleles.

By 1990, the full complexity of the HLA class I antigens was beginning to be understood. At the time new serotypes were being determined, the problem with multiple alleles for each serotype was becoming apparent by nucleotide sequencing. RFLP analysis helped determine new alleles, but sequencing was more thorough. Throughout the 1990s, PCR kits, called SSP-PCR kits were developed that allowed, at least under optimal conditions, the purification of DNA, PCR and Agarose Gel identification of alleles within an 8 hour day. Alleles that could not be clearly identified by serotype and PCR could be sequenced, allowing for the refinement of new PCR kits.

Serotypes like B*4401, B*4402, B*4403, each abundant within those with B44 serotypes could be determined with unambiguous accuracy. The molecular genetics has advanced HLA technology markedly over serotyping technology, but serotyping still survives. Serotyping can help to reveal which primers for sequencing may best work for new sequences. Serotyping had idnetified the most similar antigens that now form the HLA subgroups.