GOU VPO Tver State Medical Academy of the Ministry of Health of Russia Department of Clinical Immunology with Allergology

MAIN HISTO COMPATIBILITY COMPLEX

Teaching aid for general immunology. Tver 2008.

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Educational and methodological development for practical classes in general immunology for 5th year students of the medical and pediatric faculties, as well as for clinical residents and doctors interested in immunology.

Compiled by Associate Professor Yu.I. Budchanov.

Head of the Department, Professor A.A. Mikhailenko

© Budchanov Yu.I. 2008

Motivation Immunogenetics is a new and important branch of immunology. Knowledge of the histocompatibility system

is necessary not only in transplantology, but also in understanding the regulation of the immune response, and the interaction of cells in the immune response. The determination of HLA antigens is used in forensic medicine, population genetic studies and in the study of the gene of predisposition to diseases.

1. The student must know: A. The structure of the human HLA system.

B. HLA antigens of classes I, II and their role in intercellular interactions. B. The concepts of genotype, phenotype, haplotype.

D. Significance of HLA typing in medicine.

E. Relationship between HLA antigens and a number of human diseases. 2. The student must be able to:

Apply the acquired knowledge of immunogenetics in clinical practice.

Questions for self-preparation on the topic of the lesson:

1. The concept of genes and antigens of histocompatibility. HLA human system. Nomenclature, gene organization (genes of classes I, II, III).

2. Antigens of classes I and III, their role in intercellular interactions, in antigen presentation T-lymphocytes, in the phenomenon of double recognition.

3. The concept of HLA phenotype, genotype, haplotype. Features of inheritance.

4. Methods for research and typing of the HLA system: serological, cell-mediated, gene (polymerase chain reaction, DNA probes).

5. Practical aspects of typing HLA antigens. HLA in populations, biological significance.

6. HLA and human diseases, association mechanisms.

LITERATURE FOR SELF-EDUCATION

1. Khaitov R.M., Ignatieva G.A., Sidorovich I.G. Immunology. Norm and pathology. Textbook. - 3rd

ed., M., Medicine, 2010. - 752 p. – [p.241 - 263].

2. Khaitov R.M. Immunology: a textbook for medical students. – M.: GEOTAR-Media, 2006. - 320p. - [With. 95-102].

3. Belozerov E.S. Clinical immunology and allergology. A-Ata., 1992, p. 31-34.

4. Zaretskaya Yu.M. Clinical immunogenetics. M., 1983.

5. Methodical development. 6. Lecture.

additional literature

Konenkov V.I. Medical and ecological immunogenetics. Novosibirsk, 1999 Yarilin A.A. Fundamentals of immunology. M., 1999, p. 213-226.

Alekseev L.P., Khaitov R.M. HLA and medicine. Sat. Modern problems of allergology, immunology and immunopharmacology. M., 2001, p. 240-260.

CAN YOU ANSWER?

(Enter at home. Self-control will identify difficult questions for discussion. In class, you will check the correctness of the answers, supplement them. Try to find answers on your own and show that you can do it.)

1. In which pair of chromosomes is the major histocompatibility complex located in humans? …………….

2. Cells of what organs and tissues contain transplant cells? …………antigens

……………………………………………………………………………….……………………. .

3. What does the abbreviation HLA stand for? …………………………………………………………………………….

………………………………………………………………………………………… .

4. On what cells are antigens of the HLA system not found? ……………………….…

…………………………………………………………………………………………. .

5. What loci, subloci does the MCGS consist of: Class I ……..……… Class II ………………………………

Grade III …………………………………….. .

6. Gene products of what class of MHCs are not expressed on the cell membrane? ……………………….

7. What cells should be isolated to detect HLA class II? ………………..…………………… .

8. How are HLA antigens detected? ………………………………………………………………

………………………………………………………………………………………….. .

9. Typed patient has 6 possible antigens HLA-A, HLA-B, HLA-C. What is the name of such a situation? …………………………….

10. What histocompatibility antigen is often found in patients with ankylosing spondylitis?

…………………….. .

11. What genes are included in HLA class III? ………………………………..……………………………

…………………………………………………………………………………………… .

12. What chains make up HLA class I antigens? ………………….

13. What chains do HLA class II antigens consist of? …………………

14. Cytotoxic lymphocyte (CD8) recognizes a foreign peptide in the complex with HLA of what class?

…………………………. .

15. Th (CD4+) recognizes a foreign antigen presented by a dendritic cell or a macrophage in combination with HLA of what class? …..………

What are the possible combinations of erythrocyte antigens in a child if the isoantigenic composition

erythrocytes
Father: AO, NM, ss, dd, Cc, Ee,		and mothers: AB, MM, SS, DD, Cc, EE.
Choose the correct answer.
	AO, MN, Ss, DD, CC, EE
	AA, MM, Ss, Dd, cc, ee
	OO, NN, Ss, Dd, CC, Ee
	AB, MN, Ss, Dd, cc, EE
	AO, NN, Ss, Dd, Cc, EE
	AB, MM, SS, Dd, cc, Ee
Write another correct answer ___, ___, ___, ___, ___, ___.			Can you do more?
How many? …………. .

Reference and theoretical materials

The Major Histocompatibility Complex (MHC) is a system of genes that control the synthesis of antigens that determine tissue histocompatibility during organ transplants and induce reactions that cause transplant rejection. Surface structures of the cytomembrane of cells that induce reactions

rejection, got the name histocompatibility antigens, and the genes encoding them were called histocompatibility genes - H-genes (Histocompatibility). The discovery of histocompatibility antigens served as the basis for the development of transplantation immunology.

Subsequently, it was proved that the major histocompatibility complex is

the main genetic system that determines the functioning of the immune system,

especially the T-system of the immune system. GCGC regulates immune response,et encodes ability to recognize "one's own" and "alien", to reject foreign cells, the ability to synthesize a number of

The classical antigens of the HLA system are not detected at all in adipose tissue and on erythrocytes, as well as on neurons and trophoblast cells.

		LOCATION SCHEME OF THE HLA SYSTEM GENES
		ON CHROMOSOME 6
DP LMP TAP DQ DR		C2 Bf C4b C4a TNF

In humans, the main histocompatibility system is called the HLA system (Human Leukocyte Antigens). This is a system of genes that control the synthesis of histocompatibility antigens. It consists of three regions located on the short arm of the 6th chromosome. These regions are called: class 1, class 2, class 3 (class I, class II, class III). The region includes genes or loci. The name of each HLA gene contains the letter designation of the locus (A, B, C) and a serial number, for example: HLA-A3, HLA-B27, HLA-C2, etc. The antigens encoded by the gene also have the same designation.. At the D locus, 3 sublocuses (DP, DQ, DR) were identified. (See diagram above). There are 138 HLA antigens on the list approved by WHO. (However, the use of DNA typing, i.e. the ability to study the genes themselves, has led to the identification of more than 2000 alleles in just recent years).

Class I includes HLA - A, -B and -C loci. These three loci of the human major histocompatibility complex control the synthesis of transplantation antigens, which can be determined by serological methods (CD - Serological Determined). Molecules of HLA class I antigens consist of 2 subunits: α- and β-chains (see figure). The heavy or α-chain consists of 3 extracellular fragments - the α1, α2, and α3 domains (extracellular domains), a small region belonging to the cell membrane (transmembrane region) and an intracellular fragment (cytoplasmic region). The light chain is β2-microglobulin, non-covalently bound to the α-chain, and not bound to the cell membrane.

The α1 and α2 domains form a recess in which a peptide (antigen region) 8-10 amino acids long can be located. This depression is called peptide-binding cleft(from English cleft).

(New HLA class I antigens discovered recently include MIC and HLA-G antigens. Little is known about them at present. It should be noted that HLA-G, which is called non-classical, has only been identified

on the surface of trophoblast cells and it provides the mother with immunological tolerance to fetal antigens.)

Class 2 region (D-region) of the HLA system consists of 3 subloci: DR, DQ, DP, encoding transplantation antigens. These antigens belong to the category of antigens detected by cell-mediated methods, namely the reaction of a mixed lymphocyte culture (English mixed lymphocyte culture - MLC). More recently, the HLA-DM and -DN loci, as well as the TAP and LMP genes (not expressed on cells), have been isolated. The classic ones are DP, DQ, DR.

Presented peptide is shown in red.

Recently, antibodies have been obtained that can identify the DR and DQ antigens. Therefore, class 2 antigens are currently determined not only by cell-mediated methods, but also serologically, as well as class 1 HLA antigens.

Class 2 HLA molecules are heterodimeric glycoproteins consisting of two different α and β chains (see figure). Each chain contains 2 extracellular domains α1 and β1 at the N-terminal end, α2 and β2 (closer to the cell membrane). There are also transmembrane and cytoplasmic regions. The α1 and β1 domains form a recess that can bind peptides up to 30 amino acid residues long.

MHC-II proteins are not expressed on all cells. HLA class II molecules are present in large quantities on dendritic cells, macrophages and B-lymphocytes, i.e. on those cells that interact with helper T-lymphocytes during the immune response, using

HLA class II molecules

T-lymphocytes

significant amount

antigens of the 2nd class, but when stimulated with mitogens, IL-2

begin to express HLA class 2 molecules.

Necessary

Mark,

all 3 types of interferons

greatly enhance

expression

HLA molecules of the 1st

on the cell membrane of various cells. So

γ-interferon in

significantly enhances the expression of class 1 molecules on T- and B-lymphocytes, but also on malignant tumor cells (neuroblastoma and melanoma).

Sometimes a congenital disorder in the expression of HLA molecules of the 1st or 2nd class is found, which leads to the development of " naked lymphocyto syndrome V". Patients with such disorders suffer from insufficient immunity and often die in childhood.

The class III region contains genes whose products are directly involved in the immune response. It includes structural genes for complement components C2 and C4, Bf (properdin factor) and tumor necrosis factor-TNF (TNF) genes. This includes genes encoding the synthesis of 21hydroxylase. Thus, class 3 HLA gene products are not expressed on the cell membrane, but are in a free state.

The HLA-antigenic composition of human tissues is determined by allelic, genes related to each of the loci, i.e. one chromosome can have only one gene of each locus.

In accordance with the basic genetic patterns, each individual is a carrier no more than two alleles of each locuso and subloci (one on each of the paired autosomal chromosomes). The haplotype (a set of alleles on one chromosome) contains one allele of each of the HLA subloci. At the same time, if an individual is heterozygous for all alleles of the HLA complex, no more than twelve HLA antigens are detected in him during typing (A, B, C, DR, DQ, DP - subloci). If an individual is homozygous for some antigens, a smaller number of antigens is detected in him, but this number cannot be less than 6.

If the typed subject has the maximum possible number of HLA antigens, this is called a “full house” (“full house” of antigens).

The inheritance of HLA genes occurs according to the codominant type, in which the offspring in

The most rich in HLA antigens are lymphocytes. Therefore, the detection of these antigens is carried out on lymphocytes. ( Remember how to isolate lymphocytes from peripheral blood).

Molecules of antigens HLA-A, -B, -C make up about 1% of proteins on the surface of lymphocytes, which is approximately equal to 7 thousand molecules.

One of the most significant advances in immunology has been the discovery of the central role played by the MHC in mammals and humans in the regulation of the immune response. In strictly controlled experiments, it was shown that the same antigen causes an immune response of different heights in organisms with different genotypes, and vice versa, the same organism can be reactive to varying degrees with respect to different antigens. The genes that control this highly specific immune response are called Ir-genes (Immune response genes). They are localized in the class 2 region of the human HLA system. Ir-gene control is realized through the -T system of lymphocytes.

Central			cellular		interactions			immune		you refuse
interaction			HLA molecules,			expressed					surfaces
antigen presenting cells			representing			for recognition		alien			antigenic
peptide, and antigen-recognizing receptor - TCR (T-cell receptor)							on the surface of the T-lymphocyte
helper. At		simultaneously			recognition		alien				going on

recognition of own HLA antigens.

T-lymphocyte helper (CD4+) recognizes a foreign antigen only in the complex with surface molecules of MHC class 2 antigen-presenting cells.

Cytotoxic lymphocytes (T-effectors, CD8+) recognize an antigen

for example, of a viral nature, in combination with an HLA molecule of class I of the target cell. Exogenous antigens are represented by class II HLA molecules,

endogenous - class I molecules.

(Thus, the process of foreign recognition is limited by self HLA antigens. This is the concept of "double recognition" or "altered self recognition".)

An important role of the HLA system is also that it controls the synthesis of complement factors involved in both the classical (C2 and C4) and alternative (Bf) pathways of complement activation. Genetically determined deficiency of these complement components can predispose to infectious and autoimmune diseases.

Practical value of HLA-typing. High polymorphism makes the HLA system an excellent marker in population genetic studies and the study of genetic predisposition to diseases, but at the same time creates problems in the selection of donor–recipient pairs in organ and tissue transplantation.

Population studies conducted in many countries of the world have revealed characteristic differences in the distribution of HLA antigens in different populations. Features of the distribution of HLA-

antigens are used in genetic research to study the structure, origin and evolution of various populations. For example, the Georgian population, belonging to the southern Caucasoids, has similar features of the HLA genetic profile with the Greek, Bulgarian, and Spanish populations, indicating a common origin.

Typing of HLA antigens is widely used in forensic practice to exclude or establish paternity or kinship.

Pay attention to the connection of some diseases with the presence of one or another HLA antigen in the genotype. This is because HLA is widely used to study the genetic basis predisposition to disease. If it was not previously assumed, for example, that the disease of multiple sclerosis has a hereditary basis, now, thanks to the study of the connection with the HLA system, the fact of a hereditary predisposition is firmly established. Using

	the HLA system, for some diseases, the mode of inheritance is also determined.
For example,	ankylosing		spondylitis		autosomal dominant			inheritance,
hemochromatosis and congenital adrenal hyperplasia - autosomal recessive. Thanks very much
	associations	ankylosing		spondylitis		HLA-B27 antigen, HLA typing

used in the diagnosis of early and unclear cases of this disease. Genetic markers of insulin-dependent diabetes mellitus have been identified.

PRACTICAL WORK

Determination of HLA antigens "in donors"

Typing of tissue antigens is performed using a set of sera, consisting of 50 or more antileukocyte sera (sera of multiparous women, giving from 10 to 80% positive reactions with fetal leukocytes, or serum of volunteers immunized

human	leukocytes containing				certain SD antigens.				Serums
multiparous women, as a result of natural immunization with husband's HLA antigens during
pregnancy, contain in some cases antibodies to HLA in a sufficiently high titer.).
Serologically		antigens			histocompatibility				determine
lymphocytotoxic		test (English)		lymphocytotoxicity test).						called
micro lymphocytotoxic				use			staging			microvolume
ingredients.

Its principle is based on the interaction of HLA molecules on the surface of lymphocytes of the examined person with specific anti-HLA antibodies and complement, which leads to cell death. Cell death is determined by conventional light microscopy after staining with vital dyes.

Suspensions of lymphocytes are mixed with antiserum to a specific antigen (HLA-B8, HLA-B27, etc.), incubated for 1 hour at 25 C, complement is added and incubated again for 2 hours at 37 C, and then trypan blue or eosin is added. If an antigen corresponding to the antibodies contained in the serum is present in the lymphocytes, the antibodies in the presence of complement damage the leukocyte membrane, the dye penetrates into their cytoplasm and they stain blue or red (if eosin was used).

What cells will be stained with HLA typing?

Based on the results of typing, the degree of compatibility of the donor and recipient and the possibility of transplantation of an organ or tissue between them are established. The donor and recipient must be compatible in terms of erythrocyte antigens ABO and Rh, leukocyte antigens of the HLA system. However, in practice it is difficult to find completely compatible donor and recipient. Selection is reduced to the selection of the most suitable dono. Transplantation is possible with

incompatibility for one of the HLA antigens, but against the background of significant immunosuppression. Selection of the optimal ratio of histocompatibility antigens between the donor and the recipient significantly prolongs the life of the graft.

The lesson will demonstrate HLA plates for leukocyte typing. Recall how to obtain a pure suspension of lymphocytes from peripheral blood cells. Think about how to protect the contents of the wells from drying out during the reaction? How are serums for HLA typing obtained?

Currently, complement-fixing monoclonal antibodies (MAT) can be used for complement typing. They are used both in the microlymphocytotoxicity test and in the immunofluorescence test. Accounting for the reaction is possible both by luminescence microscopy and by using a flow cytometer.

		modern method		determination of HLA genes DNA typing. He
based on various variants of the polymerase chain reaction (PCR) and molecular hybridization.
	these methods		lies in	accumulation of necessary		analysis of significant
quantity			its polymerization and in use, complementary probes
analyzed sections of DNA. Moreover, one of the advantages of DNA typing is that it does not
the presence of viable lymphocytes is required, and the DNA of any cells is used. But
DNA can be stored for years or decades. Required for the reaction						expensive

oligonucleotide probes, primers.

The use of the molecular genetic method - DNA typing, made it possible to significantly expand the understanding of the polymorphism of the previously known genetic loci of the HLA-A, B, C, DR, DQ, DP system. In addition, new genes have been discovered, in particular TAP, DM, LMP and others. HLA class I - E, F, G, H genes have been discovered, but the function of their products is still unclear. As of December 1998, the number of identified alleles of the HLA complex genes was 942. And as of December 31, 2000, 1349 alleles were identified by molecular genetic DNA typing, and their detection continues to grow.

NEW HLA NOMENCLATURE. As already noted, HLA class 1 molecules consist of α- and β-chains. And is only polymorphicα-chain.pAllelic variants of coding genes received a four-digit name in the new nomenclature (for example, HLA-A0201 instead of the previously used designation HLA-A2, and 12 (!) New subtypes of this antigen (new allelic variants) were identified by molecular biology methods, which received the name A0201, A0202, A0203, ... to A0212). HLA-B27 has 9 allelic specificity variants, and only some of them are associated with ankylosing spondylitis (this, of course, increases their prognostic value).

Efficiency of allogeneic kidney transplantation (according to the results of annual survival in transplantation centers that have switched to donor selection based on molecular genetic

coordinating center for organ donation and the Institute of Immunology.

Even more impressive data obtained over the past 2-3 years in the course of national (primarily in the United States) and international programs for transplantation of allogeneic, "unrelated" bone marrow. Thanks to the transition of the selection of donor-recipient pairs to -DNA typing and the creation of a bank of HLA-genotyped donors, including 1.5 million people, the annual survival rate of transplanted bone marrow has been increased by 10s -20% to 70-80% (!). In turn, this led to number of bone marrow transplants from unrelated donors in the United States (which currently has the largest number of genotyped donors and recipients) from 1993 to 1997. increased more than 8 times. Stunning

The effect of unrelated bone marrow transplants is achieved solely through the selection of fully HLA compatible donor-recipient pairs by DNA typing.

The following is an excerpt from Academician R.V. Petrov's book "Me or not me: Immunological mobiles". M., 1983. - 272 p.

“... Receiving the Nobel Prize in 1930, in his solemn lecture on this subject, Karl Landsteiner said that the discovery of ever new antigens in human tissue cells would

theoretical interest. It has found, among other practical applications, forensic applications.

Imagine the following situation: it is necessary to determine the identity of a blood stain. Whose blood is it - human or animal? There is no need to explain that this situation is most often related to forensics. And the solution of the problem often becomes the answer to the main questions of the investigation. The only way to answer it is with the help of immune sera. By no means

other indicators to distinguish between the blood of a person and, for example, a dog is impossible. Microscopic or biochemical research methods are powerless.

Forensic doctors have in their arsenal a set of immune sera of various specificity: against human, horse, chicken, dog, cow, cat, etc. proteins. The spot under study is washed off, and then precipitation reactions are put. In this case, the entire set of immune sera is used. Which serum will cause precipitation, the type of animal or person belongs to the blood of the spot under study.

Let's say the forensic scientist concludes: "The knife is stained with human blood." And the murder suspect says, “Yes. But this is my blood. Not so long ago, I cut my finger with this knife. Then the examination continues. Antisera against blood groups and HLA antigens appear on the table of criminologists. And immunology again gives the exact answer: the blood belongs to the AB group, contains the M factor, Rh-negative, histocompatibility antigens such and such, etc. The situation is final

explained. The resulting characteristic completely coincides with the antigenic characteristics of the suspect's blood. Therefore, he told the truth, it is indeed his blood.

Let us dwell on one more situation, which has a great moral connotation. Imagine that a war or other disaster separated parents from their children. The children lost their names and surnames. Is it really impossible to find your child among others? After all, erythrocyte antigens and HLA are inherited. And if the father and mother do not have a factor, then the child cannot have it either. Conversely, if both parents belong to type A, then the child cannot have blood type B or AB. The same goes for HLA antigens. And with a very high degree of certainty.”

Establishing the authenticity of the remains of members of the royal family of Nicholas II was carried out in this way, using DNA typing.

for example, in England, questions of determining paternity are particularly scrupulous. But there it is most often associated not with the war. Strict laws on paternity are explained by strict laws on heirs and inheritance rights of capitals, titles, rights, privileges.

Imagine a lord declaring as his heir a young man who was not borne by his wife. Then it may be necessary to prove that the young man is his son. Or suddenly a gentleman appears, declaring himself the illegitimate son and, therefore, the heir of a millionaire. It may be true, but it may be that this gentleman is a swindler. The question is solved by the analysis of antigens of parents and children.

The distribution of HLA antigens turned out to be different in representatives of different races of nationalities. Since 1966, an intensive study of the structure of tissue compatibility antigens, initiated by WHO, has been carried out in all countries of the world. Soon the world map was covered with immunological hieroglyphs showing where and in what combination antigens are found.

HLA. Now there is perhaps no need, like Thor Heyerdahl, to equip an expedition on a reed boat to prove the migration of the population from South America to the islands of Polynesia. It is enough to look at a modern atlas of the distribution of HLA antigens and say with confidence that in both these geographical regions there are common genetic markers.

Polymorphism of classical HLA - antigens detected by serological and cell-mediated methods

Chromosomal hybridization has established that the MHC system is localized on the short arm of the 6th human autosomal chromosome, while in mice it is located on the 17th chromosome.

Rice. 1. Schematic representation of chromosome 6.

The major histocompatibility complex occupies a significant stretch of DNA, including up to 4 * 106 base pairs or about 50 genes. The main feature of the complex is significant polygenicity (the presence of several non-allelic closely linked genes, the protein products of which are structurally similar and perform identical functions) and pronounced polymorphism - the presence of many allelic forms of the same gene. All genes of the complex are inherited in a codominant manner.

Polygenicity and polymorphism (structural variability) determine the antigenic individuality of individuals of a given species.

All MHC genes are divided into three groups. Each group includes genes that control the synthesis of polypeptides of one of the three MHC classes (I, II and III) (Fig. 3.5). Between the molecules of the first two classes there are pronounced structural differences, but at the same time, according to the general plan of the structure, they are all of the same type. At the same time, no functional or structural similarity was found between the gene products of class III, on the one hand, and classes I and II, on the other hand. The group of more than 20 class III genes is generally functionally isolated - some of these genes encode, for example, proteins of the complement system (C4, C2, factor B) or molecules involved in antigen processing.

The area of ​​localization of the genes encoding the complex of mouse MHC molecules is designated as H-2, for humans - HLA.

HLA-A , HLA-B and HLA-C are chromosomal loci whose genes control the synthesis of "classical" molecules (antigens) of class I human MHC and encode the heavy chain (alpha chain). The region of these loci occupies a region longer than 1500 kb.

The synthesis of molecules (antigens) of class II human MHC is controlled by genes of the HLA-D region, which encode at least six variants of alpha and ten variants of beta chains (Fig. 3.5). These genes occupy the three loci HLA-DP, HLA-DQ and HLA-DR. Most of the class II molecules belong to the products of their expression.

In addition, the HLA-D region includes the HLA-LMP and HLA-TAP genes. Small molecular weight proteins controlled by these genes are involved in the preparation of a foreign antigen for presentation to T cells.

The genes of the human loci HLA-A, HLA-B and HLA-C encode the heavy chain (alpha chain) of the "classic" MHC class I molecules. In addition, numerous additional genes have been found outside these loci, encoding "non-classical" MHC class I molecules and located in such HLA loci as HLA-X HLA-F, HLA-E, HLA-J, HLA-H, HLA-G, HLA-F.

Molecules of the major histocompatibility complex.

The spatial organization of MHC molecules has been elucidated by X-ray diffraction analysis:

MHC class I molecules (HLA allelic variants: HLA-A , HLA-B , HLA-C) are expressed on the cell surface and are a heterodimer consisting of a single heavy alpha chain (45 kDa) non-covalently linked to single-domain beta2-microglobulin (12 kDa), which also occurs in free form in blood serum, they are called classical transplantation antigens.

The heavy chain consists of an extracellular part (forming three domains: alpha1, alpha2 and alpha3 domains), a transmembrane segment and a cytoplasmic tail domain. Each extracellular domain contains approximately 90 amino acid residues, and together they can be separated from the cell surface by treatment with papain.

The alpha2 and alpha3 domains each have one intrachain disulfide bond that loops 63 and 68 amino acid residues, respectively.

The alpha3 domain is homologous in amino acid sequence to the immunoglobulin C domains, and the conformation of the alpha3 domain resembles the folded structure of the immunoglobulin domains.

Beta2-microglobulin (beta2-m) is necessary for the expression of all MHC class I molecules and has an unchanged sequence, but in mice it occurs in two forms, differing in the replacement of one amino acid at position 85. This protein corresponds in structure to the C-domain of immunoglobulins. Beta2-microglobulin is also able to interact non-covalently with non-classical class I molecules, for example, with CD1 gene products.

Depending on the species and haplotype, the extracellular portion of class I MHC heavy chains is glycosylated to varying degrees.

The transmembrane segment of MHC class I consists of 25 predominantly hydrophobic amino acid residues and spans the lipid bilayer, most likely in an alpha-helical conformation.

The main property of class I molecules - binding peptides (antigens) and presenting them in an immunogenic form for T cells - depends on the alpha1 and alpha2 domains. These domains have significant alpha-helical regions, which, when interacting with each other, form an elongated cavity (slit) that serves as a binding site for the processed antigen. The resulting antigen complex with alpha1 and alpha2 domains determines its immunogenicity and the ability to interact with antigen-recognizing T-cell receptors.

Class I includes A antigens, AB antigens, and AC antigens.

Class I antigens are present on the surface of all nucleated cells and platelets.

Class II MHC molecules are heterodimers built from non-covalently linked heavy alpha and light beta chains.

A number of facts indicate a close similarity of alpha and beta chains in terms of their general structure. The extracellular part of each of the chains is folded into two domains (alpha1, alpha2 and beta1, beta2, respectively) and connected by a short peptide to a transmembrane segment (approximately 30 amino acid residues long). The transmembrane segment transitions into a cytoplasmic domain containing approximately 10-15 residues.

The antigen-binding region of class II MHC molecules is formed by alpha-helical regions of interacting chains similar to class I molecules, but with one significant difference: the antigen-binding cavity of class II MHC molecules is formed not by two domains of one alpha chain, but by two domains of different chains - alpha1 and beta1 domains.

The general structural similarity between the two classes of MHC molecules is evident. This is the uniformity of the spatial organization of the entire molecule, the number of domains (four), the conformational structure of the antigen-binding site.

In the structure of class II molecules, the antigen-binding cavity is more open than in class I molecules, so longer peptides can fit in it.

The most important function of class II MHC (HLA) antigens is to ensure interaction between T-lymphocytes and macrophages during the immune response. T-helper cells recognize a foreign antigen only after it has been processed by macrophages, combined with HLA class II antigens, and the appearance of this complex on the macrophage surface.

Class II antigens are present on the surface of B lymphocytes, activated T lymphocytes, monocytes, macrophages, and dendritic cells.

MHC class II genes encode membrane-bound transmembrane peptides (glycoproteins). Molecules of class II histocompatibility antigens (DR, DP, DQ), as well as class I, are heterodimeric proteins consisting of a heavy alpha chain (33 kDa) and a light beta chain (26 kDa), encoded by the genes of the HLA complex. Both chains form two domains: alpha1 and alpha2, as well as beta1 and beta2.

Class II MHC products are associated primarily with B-lymphocytes and macrophages and serve as recognition structures for T-helpers.

MHC class III genes, located within or closely linked to the MHC gene group, control several complement components: C4 and C2, as well as factor B, located in the blood plasma rather than on the cell surface. And unlike MHC class I and class II molecules, they are not involved in the control of the immune response.

The term MHC class IV is used to describe certain MHC-linked loci.

The study of the expression of MHC class I and II molecules on various cell types revealed a wider tissue distribution of class I molecules compared to class II molecules. While class I molecules are expressed on almost all studied cells, class II molecules are expressed mainly on immunocompetent cells or cells that are relatively nonspecifically involved in the formation of the immune response, such as epithelial cells.

In table. 1 presents data on the nature of the tissue distribution of MHC molecules in mice and humans.

tab. 1 Tissue distribution of MHC class I and II molecules in mice and humans

cell type	H-2 complex mice		human HLA complex
	Class I	Class II	Class I	Class II
thymocytes
macrophages
Granulocytes
Reticulocytes
red blood cells
platelets
fibroblasts
epithelial cells
epidermal cells
cardiac muscle
Skeletal muscle
Placenta
spermatozoa
Oocytes
trophoblast
Blastocytes
Embryonic tissue

The representation of class I molecules on almost all cell types correlates with the dominant role of these molecules in allogeneic graft rejection. Class II molecules are less active in the process of tissue rejection. Comparative data on the degree of participation of molecules of I and II classes of MHC in some immune responses demonstrate that some properties of MHC are more associated with one of the classes, while others are a characteristic feature of both classes (Table 2)

Tab. 2 Participation of MHC class I and II molecules in some immune responses

Charles B. Carpenter

Antigens that provide intraspecific differences in individuals are designated as alloantigens, and when they are included in the process of rejection of allogeneic tissue grafts, they become known as tissue compatibility (histocompatibility) antigens. Evolution has fixed a single region of closely linked histocompatibility genes, whose products on the cell surface provide a strong barrier to allotransplantation. The terms "major histocompatibility antigens" (major histocompatibility antigens) and "major histocompatibility gene complex" (MHC) (major histocompatibility gene complex) refer respectively to the gene products and genes of this chromosomal region. Numerous minor histocompatibility antigens, on the contrary, are encoded by multiple regions of the genome. They correspond to weaker alloantigenic differences between molecules that perform various functions. Structures carrying MHC determinants play a significant role in immunity and self-recognition during cell and tissue differentiation. Information about the MHC-control of the immune response was obtained in experiments on animals, when the immune response genes were mapped inside the MHC-in mice (H-2), rats (RT1), guinea pigs (GPLA). In humans, the MHC is named HLA. The individual letters of the abbreviation HLA are given different meanings, and with international agreement, HLA serves to designate the human MHC complex.

Several generalizations can be made about the MHC. First, in a small region (less than 2 centimorgan) MHC encodes three classes of gene products. Class I molecules, expressed by almost all cells, contain one heavy and one light polypeptide chain and are products of three reduplicated loci - HLA-A, HLA-B and HLA-C. Class II molecules, whose expression is limited to B-lymphocytes, monocytes and activated T-lymphocytes, contain two polypeptide chains (? and?) of unequal size and are products of several closely linked genes, collectively referred to as the HLA-D zone. Class III molecules are complement components C4, C2 and Bf. Second, class I and II molecules form a complex with the pseudoantigen, or the histocompatibility antigen and the pseudoantigen are recognized together by T-lymphocytes that have an appropriate receptor for the antigen. Recognition of self and non-self at the start and in the effector phase of the immune response is directly directed by molecules of classes I and II. Thirdly, there are no clear restrictions on intercellular interactions involving suppressor T-lymphocytes in humans, but the role of HLA genes is quite important for some manifestations of suppressor T-cell activity. Fourth, the MHC region contains genes for enzyme systems that are not directly related to immunity, but are important for the growth and development of the skeleton. Known HLA loci on the short arm of chromosome 6 are shown in Fig. 63-1.

Loci of the HLA system. Class I antigens. HLA class I antigens are determined serologically using human sera, mainly from multiparous women, and to a lesser extent using monoclonal antibodies. Class I antigens are present at varying densities in many body tissues, including B cells, T cells, and platelets, but not on mature red blood cells. The number of serologically detectable specificities is large, and the HLA system is the most polymorphic of the known human genetic systems. Within the HLA complex, three loci are clearly defined for serologically detectable HLA class I antigens. Each class 1 antigen contains a 2-microglobulin subunit (mol. wt. 11500) and a heavy chain (mol. wt. 44000) that carries antigenic specificity (Fig. 63-2). There are 70 well-defined A and B specificities and eight C locus specificities. The designation HLA is commonly used in the naming of major histocompatibility complex antigens, but may be omitted when the context permits. Antigens classified by the WHO inconclusively are designated with a w after the locus name. The number following the locus designation serves as the antigen's own name. The HLA antigens of the populations of Africa, Asia and Oceania are currently not well defined, although they include some of the common antigens common to people of Western European origin. The distribution of HLA antigens is different in different racial groups, and they can be used as anthropological markers in the study of diseases and migration processes.

Rice. 63-1. Schematic representation of chromosome 6.

The localization of the HLA zone in region 21 of the short arm is shown. HLA-A, HLA-B and HLA-C locals encode heavy chains of class I (44000), while? 2-microglobulin light chain (11500) class I molecules are encoded by the chromosome genome 15. The HLA-D (Class II) zone is located centrally with respect to the locals A, B with closely clutched genes of components of components 4A, C4V, BF and C2 on the site B-D. The order of the complement genes has not been established. Each class II molecule of the D-region is formed by β- and β-chains. They are present on the cell surface in different regions (DP, DQ, and DR). The digit preceding the characters? and?, means that there are different genes for chains of this type, for example, for DR there are three genes?-chains, so the expressed molecules can be 1??, 2?? or 3??. The DRw52(MT2) and DRw53(MT3) antigens are located on the 2? chain, while DR is on the l? chain. DR is non-polymorphic, while DQ antigen molecules are polymorphic in both ?- and ?-chains (2?2?). Other DQ types (1?1?) have limited polymorphism. DP polymorphism is associated with?-chains. The total length of the HLA region is about 3 cm.

Because the chromosomes are paired, each individual has up to six serologically detectable HLA-A, HLA-B, and HLA-C antigens, three from each parent. Each of these sets is designated as a haplotype, and in accordance with simple Mendelian inheritance, a quarter of the offspring have identical haplotypes, half are part of the common haplotypes, and the remaining quarter are completely incompatible (Fig. 63-3). The significance of the role of this gene complex in the transplant response is confirmed by the fact that selection of donor-recipient pairs according to the haplotype among the offspring of one generation provides the best results in kidney transplantation - about 85-90% of long-term survival (see Chapter 221).

class II antigens. The HLA-D zone is adjacent to the class I loci on the short arm of the 6th chromosome (see Fig. 63-1). This region encodes a series of class II molecules, each of which contains an ?-chain (they say, mass 29000) and ?-chain (mol. mass 34000) (see Fig. 63-2). Incompatibility in this region, especially in DR antigens, determines the proliferative response of lymphocytes in vitro. Mixed lymphocytic reaction (MLR) is assessed by the level of proliferation in mixed lymphocyte culture (MLC) and may be positive even if the HLA-A, HLA-B and HLA-C antigens are identical (see Fig. 63-3). HLA-D antigens are detected using standard stimulating lymphocytes homozygous for HLA-D and inactivated by x-rays or mitomycin C to impart a unidirectional response. There are 19 such antigens (HLA-Dwl-19) detected using homozygous typing cells.

Attempts to determine HLA-D by serological methods first made it possible to detect a series of D-linked (DR) antigens expressed on class II molecules of B lymphocytes, monocytes, and activated T lymphocytes. Then other closely linked antigenic systems were described, which received various names (MB, MT, DC, SB). The identity of individual groups of class II molecules has now been established, and the genes of the corresponding ?- and ?-chains have been isolated and sequenced. Class II gene map shown in fig. 63-1 reflects the minimum number of genes and molecular regions. Although a molecule of mass II may contain DR? from the haplotype of one of the parents, and DR? - of the other (transcomplementation), combinatorics outside each of the DP, DQ, DR regions is rare, if not impossible. DR molecules, and to some extent DQ, can serve as stimuli for primary MLR. Secondary MLR is defined as a lymphocyte-primed test (PLT) and results in 24-36 hours instead of 6-7 days for a primary response. DP alloantigens were discovered due to their ability to induce PLT stimulation, although they do not confer primary MLR. Although B-lymphocytes and activated T-lymphocytes express all three sets of class II molecules, DQ antigens are not expressed on 60-90% of DP- and DR-positive monocytes.

Rice. 63-2. Schematic representation of class I and II cell surface molecules.

Class I molecules consist of two polypeptide chains. Heavy chain with a pier. weighing 44,000 passes through the plasma membrane; its outer region consists of three domains (α1, α2, and α3) formed by disulfide bonds. Light chain with mol. with a mass of 11500 (?2-microglobulin, ?2mu) is encoded by chromosome 15 and is non-covalently linked to the heavy chain. The amino acid homology between class I molecules is 80–85%, decreasing to 50% in regions α1 and α2, which probably correspond to regions of alloantigenic polymorphism. Class II molecules are formed by two non-covalently linked polypstide chains, ?-chain with a pier. with a mass of 34000 and?-chain with a mol mass of 29000. Each chain contains two domains formed by disulfide bonds (from S. B. Carpenter, E. L. Milford, Renal Transplantation: Immunobiology in the Kidnev/Eds. B. Brenner, F. Rector, New York: Samiders, 1985).

Rice. 63-3. HLA region of chromosome 6: inheritance of HLA haplotypes. Each chromosomal segment of linked genes is designated as a haplotype, and each individual inherits one haplotype from each parent. The diagram shows the antigens A, B and C of haplotypes a and b for this hypothetical individual; below, the designations of haplotypes are disclosed in accordance with the text. If a male with an ab haplotype marries a woman with a cd haplotype, the offspring can only be of four types (in terms of HLA). If during meiosis one of the parents undergoes recombination (marked with broken lines), then this leads to the formation of an altered haplotype. The frequency of altered haplotypes in children serves as a measure of distances on the genetic hag (1% recombination frequency == 1 cM; see Fig. 63-1) (from D. B. Carpenter. Kidney International, D)78. 14.283).

Molecular genetics. Each polypeptide chain of molecules of classes I and II contains several polymorphic regions in addition to a "private" antigenic determinant, determined using antisera. The cell-mediated lympholysis (CML) assay measures the specificity of killer T cells (TK) that proliferate in MLR by testing on target cells from donors who have not provided MLR stimulating cells. The antigenic systems detected by this method show a close but incomplete correlation with "particular" class 1 antigens. Cytotoxic cell cloning has revealed a set of polymorphic target determinants on HLA molecules, some of which cannot be detected using alloantisera and monoclonal antibodies obtained by immunizing mice with human cells. Some of these reagents can be used to identify "particular" HLA determinants, while others target more "general" (sometimes called supertyping) determinants. One such system of "common" HLA-B antigens has two alleles, Bw4 and Bw6. Most "private" HLA-Bs are associated with either Bw4 or Bw6. Other systems are associated with subgroups of HLA antigens. For example, HLA-B positive heavy chains contain additional regions common to B7, B27, Bw22 and B40 or B5, B15, B18 and Bw35. There are other types of overlapping antigenic determinants, as evidenced by the reaction of monoclonal antibodies with a site common to the heavy chains of HLA-A and HLA-B. The study of the amino acid sequence and pstid maps of some HLA molecules showed that the hypervariable regions of class I antigens are concentrated in the outer β1 domain (see Fig. 63-2) and the adjacent region of the β2 domain. Variable sequences of class II molecules are different for different loci. Remarkably, class I α3 domain, class II α2 domain, and class II β2 domain, as well as a part of the T8 membrane molecule (Leu 2) involved in intercellular interactions (see Chapter 62), show significant amino acid sequence homology with immunoglobulin constant zones. This confirms the hypothesis about the evolutionary formation of a family of gene products that carry the functions of immunological recognition. In the study of HLA genomic DNA for molecules of classes I and II, typical exon-intron sequences were found, and exons were identified for signal peptides (5") of each of the domains, the transmembrane hydrophobic segment and the cytoplasmic segment (3"). cDNA probes are available for most HLA chains, and the use of enzymatic digests to assess restriction fragment length polymorphism (RFLP) status has yielded data that correlates with class 11 serologic assays in the MLR. However, the abundance (20-30) of class 1 genes makes the evaluation of polymorphism by RFLP difficult. Many of these genes are not expressed (pseudogenes), although some may correspond to additional class I loci that are only expressed on activated T cells; their functions are unknown. The development of specific tests for the HLA-A and HLA-B loci will help to understand this rather complex problem.

Complement (class III). The structural genes for the three complement components C4, C2 and Bf are present in the HLA-B-D zone (see Fig. 63-1). These are two C4 loci encoding C4A and C4B, originally described as the erythrocyte antigens Rodgers and Chido, respectively. These antigens were in fact absorbed from the plasma by C4 molecules. The other components of complement do not have a tight bond with HLA. No crossing over has been described between the C2, Bf, and C4 genes. All of them are encoded by a section between HLA-B and HLA-DR with a length of about 100 kb. There are two C2 alleles, four Bf, seven C4A and three C4B alleles, in addition, there are silent QO alleles at each locus. The exceptional polymorphism of complement histotypes (complotypes) makes this system suitable for genetic research.

Table 63-1. Most common HLA haplotins

In table. Figure 63-1 shows the four most common haplotypes found in individuals of Western European ancestry. MLR results in unrelated individuals selected for compatibility for these haplotypes are negative, while a reaction usually occurs if unrelated individuals are matched for HLA-DR and DQ compatibility only. Such identical common haplotypes may be descended unchanged from a single ancestor.

Other genes of the 6th chromosome. Deficiency of the steroid 21-hydroxylase, an autosomal recessive trait, causes the syndrome of congenital adrenal hyperplasia (chaps. 325 and 333). The gene for this enzyme is located in the HLA-B-D region. The 21-hydroxylase gene adjacent to the C4A gene is deleted in individuals suffering from the mentioned syndrome, along with C4A (C4AQO), and the HLA-B gene can be transformed with the conversion of B 13 into rare Bw47, found only in altered haplotypes. Unlike late onset HLA-linked 21-hydroxylase deficiency, congenital adrenal hyperplasia associated with 21β-hydroxylase deficiency is not HLA-linked. Several family studies have shown that idiopathic hemochromatosis, an autosomal recessive disease, is linked to HLA (see Chapter 310). Although the pathogenesis of disorders of iron absorption in the gastrointestinal tract is unknown, it has been established that the genes that modulate this process are located near the HLA-A region.

Rice. 63-4. Scheme of the relative role of HLA-A, HLA-B, HLA-C and HLA-D antigens in the initiation of the alloimmune response and in the formation of effector cells and antibodies.

Two main classes of T lymphocytes recognize antigens: Tk - precursors of cytotoxic "killer" cells and Tx helper cells that contribute to the development of a cytotoxic response. Tx also provide assistance to B-lymphocytes in the development of a "mature" IgG response. It is important to note that TK usually recognize class I antigens, while the signal for Th is generated predominantly by HLA-D, which is closely associated with class II antigens (from C. B. Carpenter. - Kidney International, 1978, 14, 283).

immune response genes. An in vitro study of the response to synthetic polypeptide antigens, hemocyanin, collagen, and tetanus toxoid revealed that the HLA-D zone is similar to the H-2 region. I in the mouse. Presentation of antigenic fragments on the surface of macrophages or other cells bearing class II molecules requires coupled recognition of the class II molecule + antigen complex by T-lymphocytes bearing the appropriate receptor(s) (see Chapter 62). The core of this “self-)-X” or “altered self” hypothesis is that the T-dependent immune response, the action of T-helpers / inducers (Tx) is carried out only if the corresponding class II determinants are synthesized. The genes of the latter are the Ir-genes. Since class I allogeneic determinants are recognized as having already been altered, allogeneic MLP is a model of the immune system in which the presence of a pseudoantigen is optional (Fig. 63-4). The effector phases of immunity require the recognition of a pseudoantigen in combination with its own structures. The latter in humans, as well as in mice, are molecules of class I histocompatibility antigens. Influenza-infected human cell lines are lysed by immune cytotoxic T-lymphocytes (TC) only if the responding and target cells are identical at the HLA-A and HLA-B loci. Allogeneic MLR also serves as a model for the formation of class I-restricted cytotoxic T-lymphocytes (see Fig. 63-4). Restriction details for various class I and II molecules and epitopes can be isolated using primed cells that have been expanded and cloned. For example, at the level of antigen-presenting cells, a given Th clone recognizes an antigenic fragment complexed with a specific region of a class II molecule via the Ti receptor. Restrictive elements for some microbial antigens are the DR and Dw alleles.

Suppression of the immune response (or, low level of response) to cedar pollen, streptococcal antigens and schistosome antigens is dominant and linked to HLA, which indicates the existence of immune suppression genes (Is). The presence of specific allelic associations of HLA with the level of the immune response was also shown, for example, for the castor bean antigen Ra5 - with DR2 and for collagen - with DR4.

Associations with diseases. If the major histocompatibility complex has an important biological function, what is that function? One hypothesis is that it plays a role in the immune surveillance of neoplastic cells that appear during an individual's lifetime. The importance of this system during pregnancy is great, since there is always tissue incompatibility between the mother and the fetus. A high degree of polymorphism may also contribute to the survival of species in the face of a huge number of microbial agents present in the environment. Self-tolerance (self-tolerance) can cross over to microbial antigens, resulting in high susceptibility leading to fatal infections, while polymorphism in the HLA system contributes to the fact that part of the population recognizes dangerous agents as foreign and includes an adequate response. These hypotheses link the role of HLA to the benefits of the system surviving under selection pressure. Each of these hypotheses has some support.

An important evidence of the role of the HLA complex in immunobiology was the discovery of a positive association of some pathological processes with HLA antigens. The study of these associations was stimulated by the discovery of immune response genes linked to the H-2 complex in mice. In table. 63-3 summarizes the most significant HLA and disease associations.

It has been established that the frequency of occurrence of HLA-B27 is increased in some rheumatic diseases, especially in ankylosing spondylitis, a disease of a clearly familial nature. The B27 antigen is present in only 7% of people of Western European origin, but it is found in 80-90% of patients with ankylosing spondylitis. In terms of relative risk, this means that this antigen is responsible for the susceptibility to the development of ankylosing spondylitis, which is 87 times higher in its carriers than in the general population. Similarly, a high degree of association with the B27 antigen in acute anterior uveitis, Reiter's syndrome, and reactive arthritis in at least three bacterial infections (yersiniosis, salmonellosis, and gonorrhea) has been shown. Although the common form of juvenile rheumatoid arthritis is also associated with B27, the type of disease with mild articular syndrome and iritis is associated with B27. In psoriatic arthritis of the central type, B27 is more common, while Bw38 is associated with both central and peripheral types. Psoriasis is associated with Cw6. Patients with degenerative arthritis or gout do not show any change in the frequency of antigens.

Most other associations with diseases are characteristic of HLA-D antigens. For example, gluten-sensitive enteropathy in children and adults is associated with the DR3 antigen (relative risk 21). The actual percentage of patients with this antigen varies from 63 to 96% compared to 22-27% in controls. The same antigen is more often found in patients with active chronic hepatitis and dermatitis herpetiformis, who also suffer from gluten-sensitive enteropathy. Juvenile insulin-dependent diabetes mellitus (type I) is associated with DR3 and DR4 and negatively associated with DR2 In 17-25% of patients with type I diabetes, a rare allele Bf (M) was found. Diabetes with onset in adulthood (type II) has no association with HLA. Hyperthyroidism in the US is associated with B8 and Dw3, while in the Japanese population it is associated with Bw35. A broader survey of healthy and sick representatives of various races will help clarify the issue of universal HLA markers. For example, the B27 antigen, which is rare in healthy Japanese individuals, is common in patients with ankylosing spondylitis. Similarly, DR4 is a marker for type I diabetes in all races. Sometimes the HLA marker is clearly associated with only a part of the symptoms within the syndrome. For example, myasthenia gravis is significantly more strongly associated with B8 and DR3 antigens in patients without thymoma, and multiple sclerosis is associated with DR2 antigen in individuals with a rapidly progressive course of the disease. Goodpasture's syndrome associated with autoimmune damage to the glomerular basement membranes, idiopathic membranous glomerulonephritis, reflecting autoimmune processes with the formation of antibodies to glomerular antigens, as well as gold-induced membranous nephritis, are largely associated with HLA-DR.

Table 63-3. Diseases associated with HLA antigens

Unbalanced grip. Although the distribution of HLA alleles varies in racial and ethnic populations, the most characteristic feature of the population genetics of HLA antigens is the presence of linkage disequilibrium for some A and B, B and C, B, D antigens and complement loci. Linkage disequilibrium means that antigens from closely linked loci are found together more often than would be expected from the assumption of random association. A classic example of linkage disequilibrium is the association of the AHLA-A1 locus antigen with the HLA-B8 B locus antigen in individuals of Western European origin. The simultaneous presence of A1 and B8, calculated on the basis of the frequencies of their genes, should be observed with a frequency of 0.17. 0.11, i.e. approximately 0.02. Whereas the observed frequency of their coexistence is 0.08, i.e., 4 times higher than expected, and the difference between these values ​​is 0.06. The last value is denoted by delta (?) and serves as a measure of nonequilibrium. Linkage disequilibrium of other A- and B-locus haplotypes was also found: A3 and B7, A2 and B12, A29 and B12, A11 and Bw35. For some D-zone determinants, linkage disequilibrium with B-locus antigens (for example, DR3 and B8) is described; as well as for antigens of B and C loci. Serologically detectable HLA antigens serve as markers for whole haplotype genes within a family and as markers for specific genes in a population, but only in the presence of linkage disequilibrium.

Linkage disequilibrium is significant because such gene associations can generate certain functions. Selection pressure during evolution may be a major factor in the maintenance of certain gene combinations in genotypes. For example, there is a theory that A1 and B8, as well as some determinants of D and other regions, provide a selective advantage in the face of epidemics of diseases such as plague or smallpox. However, it is also possible that the descendants of people who survived such epidemics remain susceptible to other diseases, because their unique gene complex does not provide an adequate response to other environmental factors. The main difficulty of this hypothesis lies in the assumption that selection acts on several genes simultaneously and thus ensures the occurrence of the observed values ​​of P, however, the need for complex interactions between the products of different loci of the MHC complex is only the initial link for the observed phenomena, and selection can enhance multiple linkage disequilibrium. The retention of some common haplotypes named above supports this view.

On the other hand, the selection hypothesis need not explain linkage disequilibrium. When a population lacking some antigens is crossed with another population characterized by a high frequency of these antigens in equilibrium, ? may appear after several generations. Like growth? for A1 and B8, found in populations from east to west, from India to Western Europe, can be explained on the basis of population migration and assimilation. In small groups, disequilibrium can be due to compatibility, founder effects, and genetic drift. Finally, some cases of linkage disequilibrium are the result of non-random crossing over during meiosis, as chromosome segments can be more or less brittle. Whether it is selection pressure or crossover constraints, linkage disequilibrium can disappear within a few generations. A large number of non-random associations exist in the HLA gene complex, and determining their causes can provide insight into the mechanisms underlying disease susceptibility.

Clutch and associations. In table. 63-2 lists diseases that serve as an example of HLA linkage, when hereditary traits are marked within the family with the corresponding haplotypes. For example, deficiency of C2, 21-hydroxylase, idiopathic hemochromatosis are inherited in a recessive manner with partial deficiency in heterozygotes. These genetic disorders are also HLA-associated and are caused by an excess of certain HLA alleles in unrelated affected individuals. C2 deficiency is usually linked to the HLA-Aw 25, B 18, B55, D/DR2 haplotypes, and in idiopathic hemochromatosis, both linkage and a strong association between HLA-A3 and B 14 are manifested. A high degree of linkage disequilibrium in this case is caused by mutations in the person who served as its source; in addition, the period of time required for the return of the gene pool to a state of equilibrium was insufficient. From this point of view, HLA genes are simple markers of linked genes. On the other hand, interaction with specific HLA alleles may be required to manifest a particular disorder. The latter hypothesis would require the recognition of a higher rate of mutations with the expression of defective genes, which occurs only under the condition of linkage with some HLA genes.

Paget's disease and spinal ataxia are HLA-linked, autosomal dominant hereditary disorders; they are found in several family members at once. Hodgkin's disease is a manifestation of an HLA-linked recessive hereditary defect. No HLA associations have been found in these diseases, suggesting an initial multiplicity of originators of these diseases with mutations associated with different HLA alleles.

Linkage to HLA is easily determined when the dominance and recessiveness of traits are easy to distinguish, i.e., when expressivity is high and the process is determined by a defect in single genes. In most associations, HLA markers reflect risk factors involved in the implementation and modulation of the immune response under the influence of multiple genes. An example of a polygenic immune disease is atonic allergy, in which association with HLA may only be apparent in individuals with low genetically controlled (not due to HLA) levels of IgE production. Another example of this kind is the IgA deficiency (see Table 63-3) associated with HLA-DR3.

Clinical significance of the HLA system. The clinical significance of HLA typing for diagnosis is limited to the determination of B27 in the diagnosis of ankylosing spondylitis; however, in this case, there are 10% of false positive and false negative results. The study of HLA is also of value in the practice of genetic counseling for the early detection of diseases in families with idiopathic hemochromatosis, congenital adrenal hyperplasia associated with steroid hydroxylase deficiency, especially if HLA typing is performed on cells obtained by amniocentesis. The high degree of polymorphism in the HLA system makes it a valuable tool for testing various cell preparations, especially in forensic medicine. Some diseases, such as type I diabetes mellitus and others, for which HLA associations are indicated, require further study of the role of the components of the HLA system in the pathogenesis of these diseases.

Genetics of the Major Histocompatibility Complex
In the 20s of the 20th century, large-scale work was carried out at the Jackson Laboratory (Bar Harbor, USA) to obtain genetically pure lines of mice through long-term inbreeding. In experiments with interlinear transplantation of tumors, employees of this laboratory J.D. Little (G.D. Little), J. Snell (G. Snell) and other American researchers have established the existence of several dozen (more than 30) genetic loci, the difference in which causes rejection of transplanted tissues. They were designated as histocompatibility loci (H-loci, from the English Histocompatibility). At the same time, the English immunologist P. Gorer solved a similar problem by studying the blood groups of mice. In 1948, in the joint work of J. Snell and P. Gorer, the histocompatibility locus was described, which determines the strongest rejection reaction. It was named H-2 because it matched the gene for the 2nd blood group of mice. The complex structure of this genetic complex, which includes a very large number of genes, was soon established. By that time, the immunological nature of transplant rejection had already been proven, and it was clear that the effect of incompatibility at H-locuses was due to differences in the antigens encoded by the genes of this locus. Such antigens became known as alloantigens, or histocompatibility antigens.
In the 1960s, the French immunohematologist J. Dausset described several leukocyte antigens similar to some H-2 allelic products. Soon, J. Dosset, together with other specialists in transplantation genetics, based on the analysis of the data accumulated by that time on human alloantigens, postulated the existence in humans of a genetic complex similar to the H-2 locus in mice. Several alloantigens, previously discovered through the use of sera from women who gave birth many times, were identified as belonging to this complex. These sera contained antibodies to fetal alloantigens. The discovered genetic complex was named HLA (for Human leukocyte antigens). Similar complexes were found in all studied mammals and birds. In this regard, a general designation for genetic complexes of this kind was introduced - MHC (from Major histocompatibility complex). This designation was also transferred to gene products - MHC antigens.
The H-2 complex is located on mouse chromosome 17; the HLA complex is in the short arm of human chromosome 6 (6p). The structure of the human HLA locus is schematically shown in fig. 3.28. It occupies a very large

Rice. 3.28. Major histocompatibility complex (MHC) gene map using the human leukocyte antigen complex (HLA) as an example. The chromosome segment is divided into 4 segments, presented in the figure sequentially. On the right are the numbers of 3'-nucleotides of each segment

space - 4 million base pairs and contains more than 200 genes. There are 3 classes of MHC genes - I, II and III. The rejection of incompatible grafts and antigen presentation to T cells involve class I and II gene products located in the 3'- and 5'-parts of the complex, respectively. Initially, they were divided according to the induction by their products of predominantly humoral (class I) or cellular (class II, described somewhat later than I) immunity. There are 2 groups of class I genes. The first is formed by genes A, B and C, which are characterized by unprecedentedly high polymorphism - several hundred of their allelic forms are known (for example, HLA-B - 830) - see table. 3.7. These are classic class I genes. Another group is formed by nonclassical genes E, F, G, H (genes with limited polymorphism). Only classical class I gene products are involved in antigen presentation to T-lymphocytes.
Table 3.7. Human leukocyte antigen (HLA) gene polymorphisms

The end of the table. 3.7

Class	Locus	Number of alleles identified by DNA typing
II	HLA-DRA	3
	HLA-DRB1	463
	HLA-DRB2-9	82
	HLA-DQA1	34
	HLA-DQB1	78
	HLA-DPA1	23
	HLA-DPB1	125
	HLA-DOA	12
	HLA-DOB	9
	HLA-DMA	4
	HLA-DMB	7
Total		2478

MHC class II genes also include several variants. The products of the DR (a and b), DP (a and b) and DQ (a and c) genes encoding the corresponding polypeptide chains of molecules are directly involved in the presentation of the antigen. In all cases, the β-chain genes are characterized by a significantly higher polymorphism than the α-chain genes. The later discovery of these genes is associated with difficulties in identifying their products: the sera of multiple parous women used to detect MHC products contained antibodies to MHC molecules almost exclusively of class I. They revealed only alloantigenic variants of the HLA-DRB gene. A mixed culture of lymphocytes (i.e., T-cell reaction) was used to determine class II molecules, providing much less opportunity to detect the subtleties of antigenic differences. Currently, antigens of both classes are determined in the polymerase chain reaction (i.e., it is the genes that are determined, and not their products, as before). Class II includes several genes with a low level of polymorphism, the products of which do not present the antigen, but are involved in its intracellular processing - processing (TAP, LMP genes) or contribute to the incorporation of the antigenic peptide into MHC-II molecules (HLA-DM, HLA-DO).
MHC class III genes, as already mentioned, are not involved in histocompatibility molecules and their presentation. They encode some complement components, tumor necrosis factor family cytokines, and heat shock proteins.
The structure of the mouse H-2 locus is similar to that of the human HLA locus described above. The main difference concerns the localization of class I (K and D) genes, which are spatially separated in mice, while the location of class II (A, E) and III genes corresponds to that in the human HLA locus.

MHC molecules are polymorphic products of major histocompatibility complex classes I and II
Despite a significant similarity in the general plan of the structure of MHC molecules of classes I and II, they have a number of differences. The scheme of the domain structure of these molecules is shown in Fig. . 3.29. Molecules of both types are formed by two polypeptide chains containing 1-3 domains (Table 3.8). Each domain contains about 90 amino acid residues. Molecules of MHC classes I and II have a similar molecular weight - about 60 kDa.

Rice. 3.29. Scheme of the structure of MHC molecules

Table 3.8. Characterization of polypeptide chains of HLA class I and II molecules

Molecule	Chain name	to her O ABOUT to her S o A	Extracellular domains	1 I O. g 1 | F Z, 2? *^th in Mr. s n *	Number S-S-ties	Number of residues in domains
						1 I ABOUT H F h f w I 3 CQ Q	1 Yu S f S « « 3 and I her sch N o.	I* A n *^m O and 2 o? n S I Y I 2
HLA class I	"1	45	ab ^ a3	There is	2	90-90-90	25	30
	v2-micro gloublin	12	v2-micro globulin	No	0	100	-	-
HLA class II	A	33-35	ai, a2	There is	1	90-90	25	varies
	V	29	Pi, v2	There is	2	90-90	25	varies

In class I molecules, the polypeptide chains are very different from each other. Chain a consists of three extracellular domains, of which the 3rd one (adjacent to the membrane) belongs to the immunoglobulin superfamily, and the other 2 have a different structure, which we will consider below. a-Chain is anchored in the membrane; in addition to transmembrane, it has a short cytoplasmic region (30 residues) that does not have enzymatic activity and is not associated with enzymes. The β-chain, also called P2-microglobulin, belongs to the immunoglobulin superfamily. It is non-covalently bound to the α3 domain of the α chain and does not have a transmembrane region. p2-Microglobulin is encoded by a gene located outside the MHC complex (on chromosome 15). The described structure is characteristic of human HLA-A, HLA-B and HLA-C molecules, as well as mouse H-2K and H-2D molecules and MHC-I molecules of all other animal species.
MHC-II molecules also have the same structure for human HLA-DP, HLA-DQ, HLA-DR, as well as mouse H-2A and H-2E. They include 2 chains of a similar structure - a and p. Both chains penetrate the membrane, have 2 domains in the extracellular part and a short (12-15 residues) cytoplasmic region. The a2 and p2 domains adjacent to the membrane belong to the immunoglobulin superfamily, while the distal aj and Pj domains are structurally similar to the a1 and a2 domains of MHC-I molecules.
Thus, all MHC molecules in total contain 2 near-membrane domains of the immunoglobulin superfamily and 2 distal domains of a different (similar) structure. The distal domains in MHC-I molecules are formed by a single chain (a), while in MHC-II molecules they are formed by different chains (a and p). It is these distal domains of MHC molecules that bind the antigenic peptide and play a key role in the formation of the TCR ligand.
Schematically, the structure of antigen-binding cavities (or grooves, crevices - from English - groove) is shown in fig. 3.30. Cavities have a bottom and walls. The bottom is a flat area lined with the p-layered (N-terminal) portion of the polypeptide chain domains, while the walls are formed by the C-terminal a-helical portions of the domains. In MHC-I molecules, this entire structure is formed by a continuous polypeptide chain of a1 and a2 domains of a single a-chain, while in MHC-II molecules, the peptide-binding cavity is formed by the domains of two different chains (a1- and Pj-domains of the corresponding chains) adjacent to each other in the region of the b-structured bottom of the groove.
Above we spoke about the extremely high polymorphism of classical MHC molecules of both classes: there are several hundred allelic variants of genes and, consequently, their protein products. If we superimpose the location of varying amino acid residues on the scheme of MHC molecules, it turns out that, firstly, they are located mainly in the distal domains (a1 and a2 - in MHC-I molecules, a1 and Pj - in MHC-II molecules), and secondly, they are associated almost exclusively with the walls of the antigen-binding cavity. In MHC-II molecules, variability predominates in the part of the walls formed by the p domain. Thus, this cavity has a standard organization, but depending on the MHC genotype, the fine details of its structure vary. The affinity of various peptides for antigen binding

Rice. 3.30. Three-dimensional models of the structure of molecules of the major histocompatibility complex. Spatial models of the molecules of the major histocompatibility complex, presented from different angles of view (according to Bjorkman et al, 1987)

gap of MHC molecules varies over a wide range. An affinity of the order of 10-5 M is considered quite high.
Let us emphasize one very important circumstance concerning the variability of key molecules of the immune system. An exceptionally high level of variability is characteristic of both antigen-recognizing structures (antibodies, TCR) and MHC molecules involved in the construction of the TCR ligand.

the level of human and animal populations, while in each particular organism there can be no more than 2 variants of molecules - products of allelic genes. If we take into account that a person has 8 highly polymorphic MHC genes (A, B, C, as well as p-genes DP, DQ and DR and a-genes DP and DQ), then the number of variants of MHC polypeptide chains cannot exceed 16.
MHC-I and MHC-II molecules are present on the cell surface, but differ significantly in tissue distribution. MHC-I molecules are present on almost all nucleated cells of the body and are absent on erythrocytes and villous trophoblast cells. Each cell usually contains about 7000 MHC-I molecules. The density of their expression can change under the influence of various factors, in particular, cytokines. MHC-II molecules are present on the surface of a limited number of cell types. They are expressed primarily on APC - dendritic cells, B-lymphocytes and activated macrophages. The content of molecules on the surface of these cells varies greatly. One dendritic cell usually contains about 100,000 MHC-II molecules. Under certain conditions (for example, during inflammation), they can appear on the surface of other activated cells - epithelial, endothelial, etc. The classic inducer of MHC-II molecules is IFNy. A feature of MHC membrane molecules is their rapid exchange on the cell surface, which is especially characteristic of MHC-I (molecule renewal time is about 6 h).
A special group of antigen-presenting molecules is formed by homologues of MHC-I products - CD1 molecules (CD1a, CD1b, CD1c and CD1d), encoded by five polymorphic genes (CD1 A-D) localized in humans on chromosome 1. In their structure, CD1 molecules are similar to MHC-I (homology is 20-25%). They have a similar domain structure (domains aj, a2, and a3). CD1 - transmembrane proteins associated with the p2-microglobulin molecule. The molecular weight of the protein part of the CDl-complex is 33 kDa. The aj and a2 domains form an antigen-binding cavity closed at both ends (as in MHC-I molecules). Its capacity is somewhat larger than in MHC-I molecules. CD1 binds bacterial and autologous lipids (diacylglycerol, mycolic acid, etc.) and lipopeptides. CD1d differs from other CD1 molecules in a number of properties. This molecule binds autologous glycolipids. Its best known ligand is a-galactosylceramide. CD1a, CD1b, and CD1c molecules are expressed on the surface of dendritic cells, monocytes, and macrophages, with CD1c serving as a marker for the entire population of dendritic cells in humans, and CD^ for Langerhans cells. CD1d is expressed in low amounts on dendritic cells (except Langerhans cells), monocytes, and macrophages.