Basic Immunology
and Disorders of The Immune System
Francisco G. La Rosa, MD

II. Basic Concepts of Inflammation

A. Phagocytosis
B. Complement
C. Cytokines:

Interleukin 1
Interleukin 2
Interleukin 4
Interleukin 6
Gamma-interferon

D. Histocompatibility system: Significance of the HLA Complex

 
A. Phagocytosis

Phagocytic cells:

1. Neutrophils circulate in the blood and migrate quickly in response to local invasion by microorganisms.

2. Monocytes are derived from bone marrow stem cells and circulate in the blood. They migrate to the tissues, where they differentiate into macrophages, which reside in all body tissues (e.g., Kupffer cells of the liver).

Phagocytic process:

1. Ameboid movement.
2. Chemotaxis.
3. Ingestion.
4. Digestion.

Lysosomal granules: The contents of lysosomal granules include two mechanisms for destroying foreign particles:

1. Certain proteins kill microorganisms by oxygen-independent mechanisms.

a. Proteinases
b. Cationic proteins
c. Lysozyme
d. Lactoferrin

2. Other microbicidal compounds are generated by oxygen-dependent mechanisms, including:

a. Myeloperoxidase
b. Hydrogen peroxide
c. Superoxide anion
d. Single oxygen
e. Hydroxyl radical

Two kinds of lysosomal granules:

1. Primary granules, azurophilic granules.
2. Secondary or specific granules, alkaline phosphatase, lactoferrin, and lysozyme.

Opsonization. Phagocytosis can be remarkably enhanced in the presence of blood serum or plasma, because of opsonization, which is the process of enhancing phagocytoses via the presence of opsonins. Molecules attached to particles serve as ligands for specific phagocyte receptors:

1. The C3b split product of the complement cascade
2. C5a and C5b67
3. Antibodies
4. Fibronectin
5. Leukotrienes
6. Tuftsin

B. Complement

The complement system plays a major role in host defense and the inflammatory process. Complement consists of a complex series of at least 15 plasma proteins that normally are functionally inactive. Activation of the complement system results in:

Opsonic function, complement components coat pathogenic organisms or immune complexes, facilitating the process of phagocytosis.

Inflammatory function, induction of histamine release from mast cells and basophils and stimulation of the inflammatory response.

Cytotoxicity by membrane attack of target cells (e.g., bacteria and tumor cells) leading to cell lysis.

B1. THE CLASSICAL PATHWAY OF ACTIVATION

Complement is activated sequentially in a cascading manner, with a protein activated only by the protein that directly preceded it in the sequence. Activation may occur via two pathways, the classical and the alternative pathways. The classical pathway require the interaction of all nine major complement components.

Activation via antigen-antibody complexes or by aggregated immunoglobulins.

1. IgG (mainly IgG1, IgG2, and IgG3) and IgM are most efficient in reacting with complement.

2. Classically, an antigen-antibody complex is designated by EA, where E is the antigen and A is the antibody. EA might represent immune complexes or antibody-coated bacteria, tumor cells, or lymphocytes. Complement components bind to EA in an orderly sequence to form a macromolecular complex, EAC 1,4,2,3,5,6,7,8,9.

The classical pathway involves the following components and steps:

1. C1: When IgG or IgM reacts with antigen, the Fc fragment of the immunoglobulin provides a C1 binding site. C1, also called the recognition unit, contains three polypeptides-C1q, C1r and C1s-that are held together by calcium ions. With the removal of the calcium, C1 breaks down into its three subunits.

a. C1q, the portion of the molecule that attaches first to immunoglobulin and initiates complement activation, has six binding sites. Because of its multivalency it can cross-link multiple immunoglobulin molecules.

b. C1q binding leads to activation of C1r proenzymes.

c. Activated C1r cleaves the proenzyme C1s. The latter acquires esterase activity and is referred to as C1 esterase (C1s). Calcium ions are essential for activation of C1.

2. C4: C1s mediates cleavage of native C4, the next component in the complement cascade, into C4a and C4b. One molecule of C1s can cleave several C4 molecules, thus serving as one of the sites in the amplification process.

a. C4a, one of three anaphylatoxins, is released into the fluid phase.

b. C4b can bind to cell membranes, but rather inefficiently.

3. C2: C1s in the presence of C4b cleaves the next component, C2, into C2a and C2b.

a. C2a remains linked to the cell-bound C4b, thus forming the bimolecular complex C4b2a. Magnesium ions are required for formation of the C4b2a complex. C4b2a has enzymatic activity and is referred to a classical pathway C3 convertase.

b. C2b is released into the fluid phase.

4. C3: The substrate for C3 convertase is C3. Circulating C3 binds to the C4b2a complex and is cleaved into two fragments, C3a and C3b.

a. C3a is an anaphylatoxin and remains unbound.

b. C3b is bound to the membrane in the vicinity of the C4b2a complex (C3 convertase), forming a trimolecular complex, C4b2a3b, which has enzymatic activity. The C4b2a3b complex is also referred to as C5 convertase, which acts on C5, the first complement component of the membrane attack pathway.

B2. THE ALTERNATIVE PATHWAY OF ACTIVATION

The alternative pathway, also referred to as the properdin pathway, is considered to be a primitive defense system, a bypass mechanism that does not require C1, C4, and C2 interaction.

Activation can be triggered immunologically (e.g., by IgA and some IgG) and non immunologically (e.g., by certain microbial cell surfaces, complex polysaccharides, and bacterial lipopolysaccharides).

The alternative pathway involves the following components and steps:

1. C3: The initial recognition event necessary for alternative pathway activation is the presence of C3, specifically C3b, which is probably continuously generated in small amounts in the circulation.

a. Factor B. C3b interacts with factor B, also called the C3 proactivator, to form C3b, B, which is a magnesium ion-dependent complex.

b. Factor D. The complex C3b, B is susceptible to enzymatic cleavage by factor D, also called C3 proactivator convertase, into two fragments, Ba and Bb. The Ba fragment is released. An active site is exposed in the Bb fragment, which remains bound to C3b, forming th C3b, Bb complex, also called amplification C3 convertase. When stabilized by the binding of properdin (P), which slows the dissociation of Bb, the C3b, Bb complex becomes a C3 convertase that cleaves C3 and generates more C3b. C3b then fixes to the activator surface so that more factor B binding sites are exposed, and the amplification loop amplifies the initial recognition event.

As more C3b is generated, the complex expands (C3bn’, Bb; where n > 1) and becomes a C5 convertase capable of cleaving C5 into C5a and C5b and initiating the membrane attack pathway.

Cobra venom factor (CVF) contained in cobra venom has the interesting property of activating the alternative complement pathway. Like C3b, it complexes with Bb to form CVF, Bb which is resistant to factors H and I and capable of continuously activating the C3 -to-C3b conversion leading to complement depletion.

B3. THE (COMMON) MEMBRANE ATTACK PATHWAY

The pathway of membrane attack, also called the common pathway, is marked by the convergence of the classical and alternative pathways at the point of C5 activation. Activation of the membrane attack complex is initiated by C5 convertase (i.e., C4b2a3b in the classical pathway and C3bn’Bb in the alternative pathway). This is the only component in the attack complex that has enzymatic activity, with cleavage occurring only once. All other components bind spontaneously.

The membrane attack complex involves the following components and steps.

1. C5 is cleaved by C5 convertase into a smaller C5a fragment and a larger C5b fragment.

a. C5a, an anaphylatoxin, is released into the surrounding fluid medium.

b. C5b is the first component of the membrane attack complex. It is the receptor for the C6 and C7 components.

2. C6 and C7: Unstable C5b binds to C6, forming a stable C5b67 complex that is bound to the target cell membrane.

3. C8 attaches to the membrane-bound C5b67 complex and membrane leakage begins. Cell lysis can occur by the C5b678 complex in the absence of C9.

4. C9 attachment to the C5b678 complex serves a primary function of greatly accelerating cytolysis by the production of circular lesions in the membrane.

5, Cell lysis. The C5b6789 complex induces the formation of hollow cylinders (tubules) about 15 nm long and 8-12 nm in diameter in the lipid bilayer of the cell membrane, allowing passage of electrolytes and water across the membrane and leading ultimately to osmotic lysis of the cell.

B4. REGULATORY MECHANISMS.

Activation of the complement components is associated with potent biological functions that, if left unchecked, would exhaust the complement system. Uncontrolled activation of the complement system is prevented by several serum proteins that bind to (inhibit) or enzymatically attack (inactivate) complement components.

B5. BIOLOGICAL CONSEQUENCES OF COMPLEMENT ACTIVATION.

During complement activation, several materials with important biological activities are generated.

C3 and C5 and their cleavage products appear to be the most important complement components in terms of biological function.

1. C3a and C5a are referred to as anaphylatoxins.

a. They cause the release of vasoactive amines (e.g., histamine) from mast cells and basophils in a manner that stimulates mediator release by IgE.

b. Mediator release causes smooth muscle contraction and increases vascular permeability, effects which can be counteracted by antihistamines and anaphylatoxin inactivator.

c. C5a is much more active than C3a on a molar basis and, in addition to anaphylatoxin activity, has a wider range of biological activity, including the following:

- Chemotactic factor
- Neutropenia
- Oxidative burst, and of degranulation of neutrophils
- Production of leukotrienes

2. C3b generation and coating on target cells through complement receptors (CR1) on monocytes, neutrophils and B cells is perhaps the major biological function of complement. Its role in the activation of the alternative complement pathway has been described. C3b also plays an important role in opsonization. C3b-coated cells also tend to aggregate (immune adherence), a process that also may promote phagocytosis.

3. C3e provokes a release of neutrophils from bone marrow causing prompt leukocytosis.

4. C3d, another cleavage product of C3b, can interact with receptors on lymphocytes.

C3 nephritic factor (C3NeF) found in the circulation of patients with mesangiocapillary glomerulonephritis. It acts as an antibody against the C3bm Bb complex and leads to a marked hypocomplementemia.

C4 and its cleavage products have certain important biological functions:

1. The binding of C1 and C4 by a virus-antibody complex can neutralize virus activity.

2. C4a, have anaphylatoxin activity, causing the release of histamine from mast cells and basophils.

3. C4b receptor sites exist on several cell types, suggesting a role for C4b in opsonization as seen with C3b.

C2 cleavage has been reported to be linked to the production of a kinin-like molecule that increases vascular permeability and contract smooth muscle. It is thought to be involved in the symptoms seen in hereditary angioedema, a disease caused by uncontrolled C1 activity due to deficiency in C1 inhibitor.

Ba and Bb. Generated exclusively by the alternative pathway have important biological functions.

1. Ba is chemotactic for neutrophils.

2. Bb activates macrophages and causes them to adhere to and spread on surfaces.

Immunodeficiencies result from a lack of complement and faulty complement activation.

C. Cytokines

Exogenous as well as endogenous agents that induce or inhibit cytokine production or action can modulate immunologic reactions. Exogenous stimuli are of primary importance as inducers of endogenous cytokines. Most cytokines are produced by many cell types in response to noxious or physiologic stimuli, whereas lymphokines are produced only by lymphocytes and have largely immunoregulatory functions.

Lymphokines that are produced largely by T cells, including IL-2, IL-4, IL-5, and IFN g, act predominantly on lymphoid cells and are immunologically induced regulators of the immune response. However, IL-2, IL-4, and IL-5 can also modulate the function of a variety of other leukocytes such as macrophages, mast cells, and eosinophils, respectively; IFN g also acts on a broad spectrum of cells in addition to lymphoid cells. In contrast, IL-3 is a lymphokine that acts as a hematopoietic growth factor. The other cytokines that modulate the activities of lymphoid and nonlymphoid cells are produced by many cell types and consist of IL-1, IL-6, IL-7, IL-8, TNF, IFN a, IFN b, and TGFb. They presumably represent intercellular signals that enable connective tissues, skin, nervous system, and other tissues to communicate with the immune system.

Cytokines, in turn, regulate each other by competition, interaction, and mutual induction in a series of lymphokine cascades and circuits with positive or negative feedback effects. For example, cytokines such as IL-1 and IL-2 induce the production of other cytokines such as TNF and g-interferon. Furthermore, IL-1 and IL-2 induce each other reciprocally. Less well known, but perhaps equally important, are observations that even mesenchymal growth factors such as TGFb can induce IL-1 production by macrophages.

In addition to cytokine regulation of cytokines, neuroendocrine hormonal peptides such as endorphins and corticosteroids, as well as products of the lipoxygenase and cyclooxygenase pathway, can have agonistic or antagonistic effects on some cytokine activities. The effects of cytokines can also be regulated at the level of cell membrane receptors. Agents that influence cytokine receptor expression modulate the activities of these mediators. Thus, a complex network of endogenous ligand-receptor interactions is involved in regulating host defense mechanisms. The therapeutic use of cytokines is still in its infancy. However, some disease states have already been shown to respond to interferons and IL-2. Agonists and antagonists of the cytokines and their receptors will probably play an important role in the eventual therapy of inflammatory, infectious, autoimmune, and neoplastic diseases.

C1. INTERLEUKIN 1 (IL-1)

Activity induced: Proliferation or differentiation of B cells; lymphokine release from activated T cells; growth of fibroblasts, synovial cells, and endothelial cells; tissue catabolism; release of prostaglandin E2, collagenase, acute phase protein; fever; natural killer cell activity; neutrophil, macrophage, lymphocyte chemotaxis.

Source: Monocytes/macrophages, dendritic cells, NKC, B-cells, T-cells, endothelial cells, fibroblasts, astrocytes, keratinocytes

C2. INTERLEUKIN 2 (IL-2)

Activity induced: T-cell growth factor. Proliferation and differentiation of T-cells; growth of activated T-cells and thymocytes; lymphokine production by T-cells; cytotoxic T-cell activity; NKC activity; lymphokine-activated killer cell activity.

Source: Activated TH1 lymphocytes, some CTLs

C3. INTERLEUKIN 3 (IL-3)

Activity induced: Growth factor for many hematopoietic cells.

Source: Activated TH1 and TH2 cells, some CTLs

C4. INTERLEUKIN 4 (IL-4)

Activity induced: Activation and growth of B cells; IgG1 and IgE switching. Growth and survival of T-cells, fetal thymocytes. Increases the growth of mast cells. Inhibits macrophage activation and helps in the formation of giant multinucleated cells. Increases class II MCH induction.

Source: Activated TH2 lymphocytes.

C5. INTERLEUKIN 6 (IL-6)

Activity induced: Growth of plasmacytomas and hybridomas; production of acute phase proteins by hepatoma cells; increased class I MHC expression on fibroblasts.

Source: T lymphocytes, monocytes, macrophages, fibroblasts, certain tumor cells.

C6. GAMMA-INTERFERON (gIF)

Activity induced: Decreases viral replication in cells inducing the production of antiviral proteins that interfere with translation of viral RNA; decreases cell growth; increases expression of class I and class II MHC molecules in macrophages, endothelial cells and parenchymal cells of various organs; increases NKC activity; increases antimicrobial and tumoricidal activity of macrophages; enhances tumor necrosis factor and lymphotoxin activity.

Source: T lymphocytes and NK cells.

D. Histocompatibility system: Significance of the HLA Complex

The mammalian immune system is a very complex network of cellular and molecular components, which are specifically encoded for by gene products. A large number of genes control specific responses to a variety of antigens, as well as cellular interactions and the transmission of antigenic specificities from generation to generation. The most important genetic component is represented by a group of genes which encode for some molecules called major histocompatibility complex (MHC) antigens. These molecules, in addition to their function in the regulation of the immune response, and in the mechanism of antigen recognition by T cells, also have a major role in the rejection of grafts and tumors. In this lecture we will review some of the most important immunogenetic aspects of tissue transplantation.

D1. MHC GENES AND MOLECULES

The major histocompatibility complex (MHC) was first described by Peter Gorer in 1936 as a blood group locus that controlled the presence of antigens on the surface of mouse erythrocytes. It was originally defined as the mouse blood group antigen-II, until it was realized that red blood cell surface antigen expression was the least significant feature of this antigenic group, and had little to do with its function. Further studies correlated these antigens with the strongest histocompatibility antigens involved in the rejection of skin and tumor grafts, and the term histocompatibility-2 (H-2) antigens was created. This gave birth to the field of transplantation immunology. Gorer's early work established for the first time that "normal and neoplastic tissues contain iso-antigenic factors which are genetically determined. Iso-antigenic factors present in the grafted tissue and absent in the host are capable of eliciting a response which results in the destruction of the graft".

For many years the correlation between MHC and graft rejection was so strong that the MHC molecules were commonly called transplantation antigens. It was only in the late sixties that the MHC was linked to the genetic control of the immune response, and as a consequence, some MHC genes were called "immune response" or Ir genes. Out of this came the terms I region to denote the location of these genes in the MHC, and I antigens (Ia) to name their molecular products. Based on structural and functional similarities, and evolutionary homologies, the MHC loci are divided into two types: class I and class II. Some confusion was created with the discovery that, in several species of vertebrates, genes coding for some complement components are intimately associated with the MHC genes (class III MHC). In humans, the class III region also includes the two structural genes for steroid 21-hydroxylase. The tumor necrosis factor genes, a and b, and lymphotoxin gene have been located between the HLA class I and III regions. However, it is now generally accepted that this genetic relationship is only due to their location on the same chromosomal segment, and has probably nothing to do with the function of the respective molecules.

1. Mouse MHC genes. The availability of inbred, congenic, and recombinant congenic strains of mice, and the production of highly specific allo-antisera to various gene products, permitted immunogeneticists to build very accurate genetic maps of the mouse MHC. Recent technology, based on DNA cloning and molecular analysis, is revealing the DNA sequence of several genes, allowing scientists to have a more detailed genetic map of the H-2 complex.

 

 

Figure 1. Simplified genetic maps of mouse and human class I and class II MHC genes. The position of the class III genes is indicated, but details of their organization have been omitted.

 

Figure 1 shows a simplified genetic map of the mouse MHC; the length of this region is approximately 1.5 centimorgans (cM) (1 cM is equivalent to a 1% recombination frequency per generation). The most extensive studies on the mouse MHC have been carried out with the BALB/c strain. Mouse class I genes are divided into two categories: genes located in the H-2 complex itself which encode for the K, D, and L molecules; and genes located within the thymus leukemia antigen (Tla) complex which encode for proteins that are structurally related to K, D, and L products, but differ in tissue distribution and, presumably, in function. The Tla complex includes the loci Qa-2,3, Tla, and Qa-1, containing more than 23 non-polymorphic genes with only a few alleles. For many years these genes were not considered to be a part of the MHC, but recent evidence indicates that they play a significant role in the immune response.

Mouse class II genes (Figure 1) encode for proteins that control mechanisms for cell-cell interaction (macrophages, thymus epithelial cells, T cells, and B cells), and the magnitude of the immune response to different antigens. These genes were originally denoted as immunoregulatory or Ir genes (I region). By functional analysis, five subregions have been mapped within the I region: I-A, I-B, I-J, I-E, and I-C. However, only the I-A and I-E subregions contain functional genes for the class II MHC molecules, the others being pseudogenes (no structural products). The composition and organization of class II genes in other experimental animals are still unknown.

2. Human MHC genes. Since the original description of leukocyte antibodies by Jean Dausset in 1952, 36 years of research have produced a considerable amount of information about the human leukocyte antigen (HLA) system. The HLA locus has been mapped on the short arm of chromosome 6 in the distal portion of the 6p 21.3 band; the b-2-microglobulin (b2m) gene is located on chromosome 15. This complex shows considerable similarity to the mouse MHC (Figure 1). The HLA complex is approximately 1.8 cM long containing approximately 3500 kilobase pairs, and it is also divided into class I and class II regions (class III region is in between these two). Class I genes code for the classical transplantation antigens HLA-A, B and C, and class II genes code for the immunoregulatory molecules (Ia equivalent) HLA-DP, DQ and DR. Unlike the detailed mapping of the mouse MHC regions, the molecular analysis of the class I HLA region has produced rather fragmentary maps.

3. Polymorphism and evolution of MHC genes. The MHC locus is one of the most highly polymorphic gene complexes known. Exchange of DNA (gene conversion) between these genes appears to be the most likely mechanism for the generation of this polymorphism. Genealogical analysis of several H-2 mutant mouse strains have indicated that at least some, if not all, of the interaction of the genes generating these mutations occurred during mitotic amplification of the germ cells. Genetic recombination among histocompatibility genes occurring in nature could readily generate mosaic transplantation genes containing sequences derived from other MHC genes. Thus, it seems likely that different forms of genetic interaction play a major role in the diversification and ongoing evolution of the MHC.

The MHC is a multigene multi-allele complex coding for glycoproteins, some of which are present on the surface of all cells. This gene complex belongs to a large family of genes that encode for other similar membrane proteins, many of them related to the immune system, e.g. immunoglobulins and T cell receptor genes (Figure 2). The MHC antigen genes of humans (HLA), mice (H-2), rats (RT), and rabbits (RLA) show close homologies between them.

4. MHC molecules. The class I and class II MHC genes encode for cell surface glycoproteins (Figure 2) which associate with foreign antigens to provide a context of self-nonself recognition by T lymphocytes. The functional structure of MHC antigens in one individual has a very close relationship with the specificity of his T cell receptor (TCR) molecules. This kinship occurs during the development of neonatal tolerance in the thymus.

Figure 2. Schematic representation of some molecules of the immunoglobulin family. Shaded areas indicate domains with high similarities in the amino acid sequence and with low degree of polymorphism even between species. Domains that are distant from the cell membrane show a high degree of polymorphism, with differences between species, individuals, and cell clones.

Class I MHC molecules represent only about 1% of the cell surface proteins, but higher levels of expression can be induced after exposure to some lymphocyte products (e.g. interferon gamma). These antigens are in very low concentration on erythrocytes, and undetectable on mature trophoblast and neurons. In humans, the HLA-C antigens are expressed in lower concentration than the HLA-A and B alleles; in some cells (e.g. platelets) the HLA-B antigens show less expression. The mouse and human class I molecules are comprised of a 45 kilo-Daltons (kDa) polypeptide (a chain) which is noncovalently associated with a lighter 12 kDa polypeptide called b-2-microglobulin (b2m). b2m is not glycosylated, contains no MHC allotypic determinants, and can also be found free as a serum protein. The heavy a chain is divided into five domains or regions: three external domains, a-1, a-2, and a-3, with 90 amino acids (aa) each, a transmembrane domain (40 aa), and the cytoplasmic domain (30 aa). The a-1 and a-2 domains contain the haplotype-determining regions and the regions that associate with antigens to interact with the TCR molecules. The a-3 domain is invariant and associates with the b2m. The complete amino acid sequence of the a-3 domain and b2m have been determined, and they show considerable homology to the constant regions of Igs.

New technological advances using recombinant DNA and gene cloning, complemented with refined biochemical analysis and X-ray crystallography, have allowed scientists to get new insights into the organization and regulation of the genetic and molecular structure of the HLA complex. Figure 3 shows a top view of the HLA-A2 molecule obtained by X-ray crystallography. The domains a-1 and a-2 form a platform composed of a single b-pleated sheet topped by a-helices with a long groove between the helices. This groove is the area where the polymorphic amino acids of class I molecules are clustered, and is the recognition site for processed foreign antigens. This site would also be the part of the molecule that carries the allotypic determinants that mediate graft rejection.

Class II MHC molecules are designated IA and IE in mice, and DP, DQ, and DR in humans. They are normally expressed on dendritic cells, some macrophages, and B cells; they are also expressed on activated human T cells. The I-E and I-A molecules are homologous to the DR and DQ molecules, respectively. The H-2 and HLA class II molecules are all composed of two glycosylated polypeptides, a and b chains, noncovalently bound (Figure 2). The a chains weigh about 33 kDa, and the b chains weigh about 28 kDa. Two carbohydrate units are bound to the a chain, and one to the b chain. Both chains contain two external domains each with 90 aa length (a-1, a-2, b-1, and b-2 respectively), a transmembrane domain of 30 aa, and a cytoplasmic domain of 10-15 aa. The allelic polymorphism is centered in the a-1 and b-1 domains. HLA-class II molecules are associated intracellularly with another transmembrane, non polymorphic, 30 kDa glycosylated polypeptide (Ii), coded by a gene in chromosome 5.

D2. HISTOCOMPATIBILITY TESTING

In order to determine the histocompatibility between two individuals, several in vivo and in vitro techniques have been designed. In transplantation research, these techniques have made it possible to study the complex immunogenetic mechanisms involved in graft rejection. In the clinical field, the goal is to find a tissue donor with the closest histocompatibility with the recipient, namely, a phenotype with the least antigenic differences with the recipient, whose tissue can survive for a long period of time with minimal immunosuppression.

1. In vitro tests. Peripheral leukocytes are the cells of choice for in vitro histocompatibility testing. They carry transplantation antigens, as well as reacting with them. When two allogeneic lymphocyte populations are mixed in the same culture (mixed leukocyte reaction or MLR), important morphological changes occur: the nucleus of the cells become euchromatic (light reticular staining) and one or more nucleoli appear, both the nucleus and the cytoplasm become larger, and the cytoplasm takes a basophilic staining quality. Under the electron microscope, the cytoplasm shows a large Golgi apparatus, abundant ribosomes and endoplasmic reticulum. The lymphocytes are now called lymphoblasts, being able to divide and proliferate as long as the stimulus and the culture conditions are appropriate.

In the original MLR tests it was difficult to study the reactivity of only one lymphocyte set, since both sets responded to each other (two way stimulation). An important improvement of this method was obtained when one cell population was prevented from replicating, without losing its capacity to stimulate the other population (one-way reaction). This was achieved by pretreatment of the stimulating population with agents that alter its DNA structure, thus suppressing replication; e.g. gamma or X-ray irradiation, nitrogen mustard, mitomycin C. Lymphocyte stimulation can be measured by changes in the cell morphology, sizing and counting of the responding cells, incorporation of 14C-thymidine, 3H thymidine, or by the detection of lymphocyte hormones (lymphokines) released to the culture medium.

2. MHC typing. Originally, class I MHC antigens were serologically identified, whereas class II antigens were recognized by MLR tests. All the tests for histocompatibility described above have been largely replaced by MHC typing techniques which use highly specific antisera (antibody typing) or lymphocytes (cellular typing) to determine the antigenic constitution of an individual. Cellular typing of the MHC remains important in the detection of HLA determinants that are still not identified by serology. Antibody and cellular MHC typing techniques are being used to assess compatibility between donors and recipients of organ and bone marrow allografts. The identification of alleles encoded within the HLA region is also used for HLA-disease association studies, paternity testing, and as a measure of immune responsiveness.

 
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