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15.4E: Histocompatibility Molecules - Biology

15.4E: Histocompatibility Molecules - Biology


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Histocompatibility molecules are glycoproteins expressed at the surface of almost all vertebrate cells. They get their name because they are responsible for the compatibility — or rather the lack of it — of the tissues of genetically different individuals. Monozygotic ("identical") human twins have the same histocompatibility molecules on their cells, and they can accept transplants of tissue from each other. The rest of us have a set of histocompatibility molecules that is probably unique to us. A graft of our tissue into another human will provoke an immune response which, if left unchecked, will end in the rejection of the transplant. So the histocompatibility molecules of one individual act as antigens when introduced into a different individual. In fact, the histocompatibility molecules are often called histocompatibility antigens or transplantation antigens.

The most rapid and severe rejection of foreign tissue occurs when there is a failure to properly match the donor and recipient for the major histocompatibility molecules. There are two categories: class I and class II.

Class I Histocompatibility Molecules

Class I molecules consist of two polypeptide chains, a long one (on the left) of 346 amino acids — it is called the heavy chain — and a short one (on the right) of 99 amino acids. The heavy chain consists of 5 main regions or domains:

  • three extracellular domains, designated here as N (includes the N terminal), C1, and C2
  • a transmembrane domain where the polypeptide chain passes through the plasma membrane of the cell
  • a cytoplasmic domain (with the C terminal) within the cytoplasm of the cell

he above figure shows a protein molecule called beta-2 microglobulin ("β2M"). It is not attached to the heavy chain by any covalent bonds, but rather by a number of noncovalent interactions. The dark bars represent disulfide (S-S) bridges linking portions of each domain (except the N domain). However, the bonds in S-S bridges are no longer than any other covalent bond, so if this molecule could be viewed in its actual tertiary (3D) configuration, we would find that the portions of the polypeptide chains containing the linked Cys are actually close together. The outermost domains ("N" and "C1") contain two segments of alpha helix that form two ridges with a groove between them. A small molecule (e.g., a short peptide) is attached noncovalently in the groove between the two alpha helices, rather like a hot dog in a bun(See fig. 15.4.5.2)

The two objects on the left of the image that look like candelabra represent the short, branched chains of sugars in this glycoprotein. The regions marked "Papain" represent the places on the heavy chain that are attacked by the proteinase papain (and made it possible to release the extracellular domains from the plasma membrane for easier analysis). The image represents the structure of a class I histocompatibility molecule, called H-2K. Almost all the cells of an animal's body (in this case, a mouse) have thousands of these molecules present in their plasma membrane. These molecules provide tissue identity and serve as major targets in the rejection of transplanted tissue and organs. But tissue rejection is not their natural function. Class I molecules serve to display antigens on the surface of the cell so that they can be "recognized" by T cells.

Humans synthesize three different types of class I molecules designated HLA-A, HLA-B, and HLA-C. (HLA stands for human leukocyte antigen; because the molecules were first studied on leukocytes). These differ only in their heavy chain, all sharing the same type of beta-2 microglobulin. The genes encoding the different heavy chains are clustered on chromosome 6 in the major histocompatibility complex (MHC). We inherit a gene for each of the three types of heavy chain from each parent so it is possible, in fact common, to express two allelic versions of each type. Thus a person heterozygous for HLA-A, HLA-B, and HLA-C expresses six different class I proteins. These are synthesized and displayed by most of the cells of the body (except those of the central nervous system).

Histocompatibility molecules present antigens to T cells

Although histocompatibility molecules were discovered because of the crucial role they play in graft rejection, clearly evolution did not give vertebrates these molecules for that function. So what is their normal function? The answer: to display antigens so that they can be "seen" by T lymphocytes. The antigen receptor on T lymphocytes (or T cells, as they are commonly called) "sees" an epitope that is a mosaic of the small molecule in the groove and portions of the alpha helices flanking it.

The small molecules ("hot dogs") are enormously diverse. They probably represent fragments derived from all the proteins present within the cell. These would include fragments of normal cell constituents, fragments of proteins encoded by intracellular parasites (like viruses), and fragments of proteins encoded by mutated genes in cancer cells.

Class II Histocompatibility Molecules

Human class II molecules are designated HLA-D, and the genes encoding them are also located in the major histocompatibility complex (MHC). Class II molecules consist of two transmembrane polypeptides. These interact to form a groove at their outer end which, like class I molecules, always contains a fragment of antigen. But the fragments bound to class II molecules are derived from antigens that the cell has taken in from its surroundings. Extracellular molecules are engulfed by endocytosis. The endosomes fuse with lysosomes and their contents are partially digested. The resulting fragments are placed in class II molecules and returned to the cell surface.

Class II molecules, in contrast to class I, are normally expressed on only certain types of cells. These are cells like macrophages and B lymphocytes that specialize in processing and presenting extracellular antigens to T lymphocytes. Thus antigen presentation by class II molecules differs from that by class I in two important ways:

  • All cells can present antigens with class I molecules, whereas only certain cells can do so with class II.
  • The antigen fragments (hot dogs) displayed in class I molecules are generated from macromolecules synthesized within the cell, whereas those displayed in class II molecules have been acquired from outside the cell.

Class I and Class II molecules present antigen fragments to different subsets of T cells

Most of the T cells of the body belong to one of two distinct subsets: CD4+ or CD8+. CD4 and CD8 are surface glycoproteins. Both CD4+ and CD8+ T cells have an antigen receptor (TCR) that "sees" a complex hot-dog-in-bun epitope. The CD8 molecules on CD8+ T cells bind to a site found only on class I histocompatibility molecules (shown here as a gray hemisphere). The CD4 molecules on CD4+ T cells bind to a site found only on class II histocompatibility molecules (shown below as a yellow triangle). However, neither type can be activated by simply binding its complementary epitope. Additional molecular interactions must take place.

The CD8+ T Cell/Class I Interaction

Because of the need for CD8 to bind to a receptor site found only on class I histocompatibility molecules, CD8+ T cells are only able to respond to antigens presented by class I molecules. Most CD8+ T cells are cytotoxic T cells (CTLs). They contain the machinery for destroying cells whose class I epitope they recognize.

Example

Every time you get a viral infection, say influenza (flu), the virus invades certain cells of your body. Once inside, the virus subverts the metabolism of the cell to make more virus. This involves synthesizing molecules encoded by the viral genome. In due course, these are assembled into a fresh crop of virus particles that leave the cell (often killing it in the process) and spread to new cells. Except while in transit from their old home to their new, the virus works inside cells safe from any antibodies. But early in their intracellular life, infected cells display fragments of the viral proteins being synthesized in the cytoplasm in their surface class I molecules. Any cytotoxic T cells specific for that antigen will bind to the infected cell and often will be able to destroy it before it can release a fresh crop of virus.

The bottom line: the function of the body's CD8+ T cells is to monitor all the cells of the body ready to destroy any that express foreign antigen fragments in their class I molecules.

The CD4+ T Cell/Class II Interaction

The CD4 molecules expressed on the surface of CD4+ T cells enable them to bind to cells presenting antigen fragments in class II molecules but not in class I. Only certain types of cells, those specialized for taking up antigen from extracellular fluids, express class II molecules. Among the most important of these are dendritic cells, macrophages (phagocytic cells that develop from monocytes that have migrated from the blood to the tissues), and B lymphocytes ("B cells") that take up exogenous antigen by receptor-mediated endocytosis.

So CD4+ T cells see antigen derived from extracellular fluids and processed by specialized antigen-presenting cells. To respond to an antigen, a CD4+ T cell must have a T cell receptor (TCR) able to recognize (bind to) a complex epitope comprising an antigenic fragment displayed by a class II molecule, bind a site on the class II molecule (shown above as a yellow triangle) with its CD4, and bind to costimulatory molecules on the antigen-presenting cell. If these conditions are met, the T cell becomes activated. Activated T cells enter the cell cycle leading to the growth of a clone of identical T cells and begin to secrete lymphokines.

Lymphokines activate and recruit other cells (e.g., mast cells) to the region producing inflammation (e.g., to cope with a bacterial infection). They also activate B cells enabling them to develop into a clone of antibody-secreting cells. The CD4+ T cells that activate B cells are called Helper T cells.


Major Histocompatibility Complex (MHC)- Types and Pathways

The major histocompatibility complex can be defined as a tightly linked cluster of genes whose products play an important role in intercellular recognition and in discrimination between self and non-self. The term ‘histo’ stands for tissue and ‘compatibility’ refers to ‘getting along or agreeable’. On the other hand, the term ‘complex’ refers to the ‘genes that are localized to a large genetic region containing multiple loci’. These genes code for antigens which involve in the determination of the compatibility of the transplanted tissue. The compatible tissues will be accepted by the immune system while the histo-incompatible ones are rejected. The rejection of foreign tissue leads to an immune response to cell surface molecules. The concept was first identified by Peter Gorer and George Snell. The main function of MHC molecules is to bring antigen to the cell surface for recognition by T cells. In humans, the genes coding for MHC molecules are found in the short arm of chromosome 6.


Chemical biology of antigen presentation by MHC molecules

MHC class I and MHC class II molecules present peptides to the immune system to drive proper T cell responses. Pharmacological modulation of T-cell responses can offer treatment options for a range of immune-related diseases. Pharmacological downregulation of MHC molecules may find application in treatment of auto-immunity and transplantation rejection while pharmacological activation of antigen presentation would support immune responses to infection and cancer. Since the cell biology of MHC class I and MHC class II antigen presentation is understood in great detail, many potential targets for manipulation have been defined over the years. Here, we discuss how antigen presentation by MHC molecules can be modulated by pharmacological agents and how chemistry can further support the study of antigen presentation in general. The chemical biology of antigen presentation by MHC molecules shows surprising options for immune modulation and the development of future therapies.


Soluble major histocompatibility complex molecules in immune regulation: highlighting class II antigens

The involvement of major histocompatibility complex (MHC) antigens in the development and regulation of immune response has been well defined over the years, starting from maturation, antigenic peptide loading, migration to the cell membrane for recognition by the T-cell receptor and recycling for immune response cessation. During this intracellular trafficking, MHC antigens find a way to be excreted by the cells, because they can be found as soluble MHC class I (sMHC-I) and class II (sMHC-II) molecules in all body fluids. Although secretion mechanisms have not been sufficiently studied, sMHC molecules have been shown to display important immunoregulatory properties. Their levels in the serum have been shown to be altered in a variety of diseases, including viral infections, inflammation, autoimmunities and cancer, etc. while they seem to be involved in a number of physiological reactions, including maintenance of tolerance, reproduction, as well as mate choice vis-à-vis species evolution. The present review aims to present the thus far existing literature on sMHC molecules and point out the importance of these molecules in the maintenance of immune homeostasis.

Keywords: major histocompatibility complex soluble class I antigens soluble class II antigens.


Plan of Action

A successful immune response to invaders requires

Activation and mobilization

Recognition

To be able to destroy invaders, the immune system must first recognize them. That is, the immune system must be able to distinguish what is nonself (foreign) from what is self. The immune system can make this distinction because all cells have identification molecules (antigens) on their surface. Microorganisms are recognized because the identification molecules on their surface are foreign.

In people, the most important self-identification molecules are called

Human leukocyte antigens (HLA), or the major histocompatibility complex (MHC)

HLA molecules are called antigens because if transplanted, as in a kidney or skin graft, they can provoke an immune response in another person (normally, they do not provoke an immune response in the person who has them). Each person has an almost unique combination of HLAs. Each person’s immune system normally recognizes this unique combination as self. A cell with molecules on its surface that are not identical to those on the body’s own cells is identified as being foreign. The immune system then attacks that cell. Such a cell may be a cell from transplanted tissue or one of the body’s cells that has been infected by an invading microorganism or altered by cancer. (HLA molecules are what doctors try to match when a person needs an organ transplant.)

T cells (T lymphocytes), as part of the immune surveillance system, must be able to recognize substances that do not belong to the body (foreign antigens). However, they cannot directly recognize an antigen. They need the help of an antigen-presenting cell (such as a macrophage or dendritic cell).

The antigen-presenting cell engulfs the antigen. Then enzymes in the cell break the antigen into fragments, which are combined with the cell's identification molecules—called major histocompatibility complex molecules, or human leukocyte antigens (HLAs). The combined HLA and antigen fragment moves to the surface of the antigen-presenting cell where it is recognized by receptors on the T cell.

Some white blood cells—B cells (B lymphocytes)—can recognize invaders directly. But others—T cells (T lymphocytes)—need help from cells called antigen-presenting cells:

Antigen-presenting cells ingest an invader and break it into fragments.

The antigen-presenting cell then combines antigen fragments from the invader with the cell's own HLA molecules.

The combination of antigen fragments and HLA molecules is moved to the cell’s surface.

A T cell with a matching receptor on its surface can attach to part of the HLA molecule presenting the antigen fragment, as a key fits into a lock.

The T cell is then activated and begins fighting the invaders that have that antigen.

How T Cells Recognize Antigens

T cells are part of the immune surveillance system. They travel through the bloodstream and lymphatic system. When they reach the lymph nodes or another secondary lymphoid organ, they look for foreign substances (antigens) in the body. However, before they can fully recognize and respond to a foreign antigen, the antigen must be processed and presented to the T cell by another white blood cell, called an antigen-presenting cell. Antigen-presenting cells consist of dendritic cells (which are the most effective), macrophages, and B cells.

Activation and mobilization

White blood cells are activated when they recognize invaders. For example, when the antigen-presenting cell presents antigen fragments bound to HLA to a T cell, the T cell attaches to the fragments and is activated. B cells can be activated directly by invaders. Once activated, white blood cells ingest or kill the invader or do both. Usually, more than one type of white blood cell is needed to kill an invader.

Immune cells, such as macrophages and activated T cells, release substances that attract other immune cells to the trouble spot, thus mobilizing defenses. The invader itself may release substances that attract immune cells.

Regulation

The immune response must be regulated to prevent extensive damage to the body, as occurs in autoimmune disorders. Regulatory (suppressor) T cells help control the response by secreting cytokines (chemical messengers of the immune system) that inhibit immune responses. These cells prevent the immune response from continuing indefinitely.

Resolution

Resolution involves confining the invader and eliminating it from the body. After the invader is eliminated, most white blood cells self-destruct and are ingested. Those that are spared are called memory cells. The body retains memory cells, which are part of acquired immunity, to remember specific invaders and respond more vigorously to them at the next encounter.

There are two major classes of lymphocytes involved with specific defenses: B cells and T cells.

Immature T cells are produced in the bone marrow, but they subsequently migrate to the thymus, where they mature and develop the ability to recognize specific antigens. T cells are responsible for cell-mediated immunity.

B cells, which mature in the bone marrow, are responsible for antibody-mediated immunity.

The cell-mediated response begins when a pathogen is engulfed by an antigen-presenting cell, in this case a macrophage. After the microbe is broken down by lysosomal enzymes, antigenic fragments are displayed with MHC molecules on the surface of the macrophage.

T cells recognize the combination of the MHC molecule and an antigenic fragment and are activated to multiply rapidly into an army of specialized T cells.

One member of this army is the cytotoxic T cell. Cytotoxic T cells recognize and destroy foreign cells and tissues or virus-infected cells.

Another T cell is the memory cytotoxic T lymphocyte, which remains in reserve in the body. If, sometime in the future, these T cells re-encounter this specific antigen, they will rapidly differentiate into cytotoxic T cells, providing a speedy and effective defense.

Helper T cells coordinate specific and nonspecific defenses. In large part by releasing chemicals that stimulate T cell and B cell growth and differentiation.

Suppressor T cells inhibit the immune response so that it ends when the infection has been controlled. Whereas the number of helper T cells increases almost at once, the number of suppressor T cells increases slowly, allowing time for an effective first response.


15.4E: Histocompatibility Molecules - Biology

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Difference Between MHC and HLA

Both are groups of antigens or proteins found on the surface of cells and in the genetic makeup or DNA. Their functions are also very similar – they identify and prevent a foreign protein or cell from entering or spreading in an organism’s body. This often happens in coordination with the immune system, which attacks these foreign bodies. Both groups of proteins regulate the immune system itself as well as its response.

The main difference between the two groups is that MHC is often found in vertebrates, while HLA is only found in humans. To simplify, HLA is the human body’s version of MHC. Part of the responsibility of these antigens is to detect cells entering the body. Upon detection, cells are recognized as either local or foreign. Local cells carrying viruses and other harmful organisms are often identified and attacked. This is also true for foreign cells introduced to the body.

These antigens are often involved when an organ transplant is planned for an organism or a human being. Certain tests are carried out to determine the compatibility between an organ and a recipient’s body. Near perfect or perfect matches are desirable in these situations in order to lessen the risk of the recipient’s body rejecting the organ.

Aside from organ transplants, both MHC and HLA are very useful in strengthening a body and its immune system. In humans, the HLA is also used in paternity tests to determine the parentage of a child this is done by comparing antigens from the child, father, and mother.

A disadvantage of MHC and HLA is that they carry certain hereditary diseases like cancer, diabetes, and lupus.

Both antigens are also responsible for preventing inbreeding or the state of excessive similar genetic material in a person. They favor diversity in the genetic makeup but, at the same time, are responsible for cooperation in terms of kin recognition, dual recognition, and transplant matching.

Both MHC and HLA have four classifications of antigens. However, only the first and second classes of antigens are responsible for identification and a response to any cell, whether local or foreign. Class I antigens deal with the destruction of foreign or infected local cells this occurs in all types of cells except for red blood cells.

Meanwhile, class II antigens mediate specific immunization to the antigen. Class II antigens are found in B cells, macrophages, and antigen-presenting cells (APCs).
MHC and HLA both act as shields of defense and protection of an organism’s body.

Summary:

1.MHC and HLA are slightly different, but their functions are basically the same.
2.Both MHC and HLA are classified as proteins and antigens. They are both located in an organism’s cells and work hand-in-hand with the body’s immune system.
3.MHC is found in many vertebrates, while HLA is only found in humans HLA is basically the human MHC.
4.Both MHC and HLA are identifiers of local and foreign cells in a body. Foreign and infected cells are attacked and immunized. MHC and HLA regulate the immune system and its responses.
5.These antigens play a key role in organ transplants an organ can be rejected by the recipient’s body if its MHC or HLA is not a near or perfect match. Aside from immunization and histocompatibility, these antigens are also responsible for a body’s defense against foreign organisms.
6.Only two classes out of four are responsible for the body’s immunity responses.
7.HLA can be used in identifying a child’s father and can also function as a carrier of hereditary diseases. It also prevents inbreeding among people.


Fusion with Lysosomes

Some pathogens have counterstrategies to avoid destruction by phagocytes. These include mechanisms to inhibit fusion of phagosomes with lysosomes, to resist the low pH environment of the lysosome, and to escape to the cytoplasm by lysing the phagolysosome membrane (Table 22-1). For example, in tuberculosis, macrophages in the lung phagocytose the bacterium Mycobacterium tuberculosis, but the bacterium evades destruction by secreting a phosphatase that dephosphorylates phosphatidylinositol (3P) and thus halts phagosome maturation.

“Escape”
Secretion of toxins that disrupt phagosomal membrane (Shigella flexneri, Listeria monocytogenes, Rickettsia rickettsii)
“Dodge”
Entrance through alternative, pathogen-specific pathway (Salmonella typhimurium, Legionella pneumophila, Chlamydia trachomatis)
Inhibition of phagosome-lysosome fusion (S. typhimurium, Mycobacterium tuberculosis)
Inhibition of phagolysosome acidification (Mycobacterium species)
“Stand and Fight”
Low pH-dependent replication (Coxiella burnetii, S. typhimurium)
Enhancement of DNA repair to survive oxidative stress (S. typhimurium)
Protective pathogen-specific virulence factors (C. burnetii, S. typhimurium)
Prevention of the processing and presentation of bacterial antigens (S. typhimurium)


RESULTS

Differential Expression of CD1b and MHC Class II in Lysosomal Subdomains of Immature DCs

MHC class II and CD1b molecules are both prominently expressed in a specialized lysosomal MIIC and mediate presentation of endocytosed antigens. However, given the distinct nature of antigens they bind, we hypothesized that some parts of the intracellular pathways for MHC class II- and CD1b-mediated antigen presentation might differ. Therefore, we carefully analyzed and quantified the MIIC in ultrathin cryosectioned human DCs by immunogold-labeled transmission electron microscopy for MHC class II and CD1b expression. When human peripheral blood monocytes were stimulated with GM-CSF and IL-4, the cells differentiated into IMDCs, and a large quantity of MHC class II molecules was detected intracellularly in the MIIC. Unlike the multivesicular structure often seen in B cells and murine DC cell lines, the MIIC developed in freshly isolated human DCs typically contained numerous membrane lamellae closely packed in concentric arrangement, and MHC class II molecules were mainly detected on internal membranes of the MIIC rather than on the limiting membrane. Besides MHC class II, these human monocyte-derived DCs also expressed CD1b molecules in the same MIIC, but unlike MHC class II, CD1b molecules seemed to be differentially localized to the limiting membrane (Figure 1A). To appreciate the differential expression of MHC class II and CD1b molecules on the inner and limiting membranes in a quantitative way, lysosomes of monocyte-derived DCs obtained from different donors were randomly analyzed and gold particles present on the internal and limiting membranes were counted for MHC class II and CD1b. As summarized in the Table 2, 85% of CD1b molecules expressed in the MIIC were found on the limiting membrane, whereas 15% were detected on the internal membranes. In sharp contrast, only 5% of MHC class II molecules expressed in the MIIC were found on the limiting membrane, and the majority of the molecules was distributed to the internal membranes. For comparison, other proteins that resided in the MIIC were also analyzed for their differential expression in the internal and the limiting membranes. As shown in Table 2, LAMP1 was localized preferentially to the limiting membrane, albeit less prominently as CD1b, whereas CD63 was expressed more abundantly in the inner membranes than in the limiting membrane.

Figure 1. After maturation, CD1b localization remains lysosomal and separates from MHC class II. Immunogold labeling of CD1b (closed arrowheads) and MHC class II (open arrowheads) on ultrathin sections of IMDC (A), 8-h LPS-stimulated DCs (B), and 48-h LPS-stimulated MDC (C). In MIICs of IMDCs (A), CD1b molecules (10-nm gold) are located at the limiting membrane (see Table 2) and MHC class II molecules (15-nm gold) are present at the internal membranes. After 8 h of maturation (B), the multilamellar structures are altering and often CD1b (10-nm gold) is no longer colocalizing with MHC class II (15-nm gold). At 48 h after maturation (C), most MHC class II is present on the plasma membrane (15-nm gold open arrowheads), and CD1b (10-nm gold) is detected on the plasma membrane, early endosomes, and electron-dense single membrane MDLs (indicated by asterisks). Scale, 200 nm. G, Golgi complex m, mitochondria p, plasma membrane and e, endosomes.

Table 2. Distribution of transmembrane proteins on the limiting membranes relative to the internal membranes of MIICs from IMDC

The immunogold labeling on multilaminar lysosomal MIICs from immature monocyte-derived dendritic cells was determined and the percentage of labeling on the limiting membranes and the internal membranes was quantified (N is the number of gold particles counted). MHC class II and CD63 molecules were detected mostly on the internal membranes and CD1b and LAMP1 on the limiting membranes. CD1c is nearly equally distributed amongst both membrane categories.

Segregation of CD1b and MHC Class II Molecules in Maturing DCs

DCs undergo dynamic cellular changes upon exposure to bacterial products, such as LPS. These changes, collectively called DC maturation, include efficient transport of peptide antigen-loaded MHC class II molecules from the lysosomal MIIC to the plasma membrane. DC maturation-associated changes in lysosomal morphology has been studied extensively at the ultrastructural level, by using a LPS-stimulated murine DC cell line, in which tubulization of the multivesicular MIIC extends toward the plasma membrane (Kleijmeer et al., 2001). However, these murine cells lack the expression of group 1 CD1 molecules and thus, it remained to be determined how CD1b and MHC class II molecules might be differentially transported from lysosomes in maturing DCs.

To investigate morphological changes in the multilamellar MIIC of maturing human DCs, monocyte-derived immature DCs were stimulated with LPS and harvested after 8 h for transmission electron microscopy. At this time point, the membrane lamellae tightly packed in concentric arrangement were appreciably loosened, and some membranes spread out and bud off to form electron-dense tubulo/vesicular structures that contained CD1b molecules, but often lacked the expression of MHC class II molecules (Figure 1B). MHC class II molecules tended to be excluded from the newly formed CD1b-containing vesicles, resulting in partial segregation of CD1b and MHC class II in these lysosomal compartments.

Distinct Steady-State Localization of CD1b and MHC Class II in Fully Matured DCs

To evaluate late morphological changes in the MIIC in fully MDCs, IMDCs were stimulated with LPS and harvested after 24 h for transmission electron microscopy. At this time point, the internal membranes of the lysosomes disappeared and the structure now was filled with electron-dense proteinaceous content. The limiting membrane still contains CD1b, whereas virtually no MHC class II molecules were detected (Figures 1C and 2). Considering the major ultrastructural changes and the changes in the presence of resident molecules, we herein propose to denote the altered mature DC lysosome MDL. The limiting membrane of the MDL contained lysosomal resident molecules LAMP1 (Figure 4). Less than 2% of the CD1b-containing compartments seemed to be late endosomes as was shown by double labeling with late endosomal marker M6PR (our unpublished data). We therefore concluded that the MDL represents a subclass of lysosomal compartments.

Figure 2. Maturation of DCs causes segregation of CD1b and CD1c from MHC class II molecules. MHC class II was detected together with CD1a (a and b), CD1b (c and d), or CD1c (e and f) by immunofluorescence on semithin cryosections of 280 nm IMDCs cultured in GM-CSF, IL-4 (IMDC a, c, and e) and after maturation with LPS (MDC b, d, and f). The CD1 isoforms are presented in the left panel, MHC class II in the middle panel, and merge of the pictures is shown in right panel. After maturation, CD1b and CD1c are located intracellular, and MHC class II molecules are located to the plasma membrane.

Figure 4. Localization of lysosomal markers CD63 and LAMP1 indicate that the electron-dense MDLs are lysosomal. In IMDCs (A), LAMP1 (15-nm gold) is mostly present on the limiting membrane of the multilamellar lysosomes, whereas CD63 (10-nm gold) can be detected on both internal and limiting membranes. On maturation with LPS (B), both LAMP1 (15 nm) and CD63 (10 nm, arrowheads) can be detected on the membrane of electron-dense structures with altered ultrastructure lacking the internal membrane structures and denoted MDLs and indicated by asterisks. Scale, 200 nm. G, Golgi complex e, endosomes m, mitochondria p, plasma membrane.

Cytoplasmic Tail Determines Internal and Limiting Membrane Lysosomal Localization of CD1b, CD1c, and CD63

In IMDCs, the localization of CD1b is on the limiting membrane of the lysosomal MIIC, whereas the MHC class II is primarily on the internal membranes. This difference in localization within the lysosome might determine the localization after maturation in the MDL. Because the internal membranes are lost in MDL, it is possible that only the molecules that form the limiting membrane of the lysosomes in IMDC are present in the MDL. Other lysosomal residents such as CD63 and LAMP1 seem to be localized preferentially to the internal and the limiting membranes of MIICs, respectively (Table 2). In contrast to CD1b, CD63, and LAMP1, CD1c molecules are almost equally distributed over the internal and limiting membrane domains. The specific enrichment of molecules on the limiting membrane could be caused by the targeting information present in their cytoplasmic tails. CD1b, CD1c, LAMP1, and CD63 all have similar tyrosine motifs in their cytoplasmic tails that is believed to target these molecules to lysosomes (Table 1). It is unknown whether the cytoplasmic motif also determines the microanatomic localization within the lysosome. In contrast to the tyrosine motifs, the lysosomal targeting of MHC class II is dependent on several factors, including the dileucine targeting signal on the beta chain and of the invariant chain. Herein, we analyzed the role of the CD1b cytoplasmic tail in sublocalization in lysosomes. First, we generated chimeras of CD1b, in which the cytoplasmic tail is exchanged for the tail of CD63, which preferentially localizes to the internal membranes of the MIIC. Also, tail chimeras of CD1b with CD1a and CD1c were evaluated to determine whether CD1b chimera's remained at the limiting membrane. As a control, we determined the localization of endogenous CD63.

The results showed that the localization of CD1b wild-type and endogenous CD63 is comparable with the distribution determined in IMDC. CD1b molecules with the CD1b tail are targeted to the limiting membrane of the lysosomes. Both the CD63 and the CD1c tail chimeric constructs target CD1b more prominently to the internal membranes (Figure 3). The CD1b/CD1a tail chimera was found in early endosomal compartments and not in lysosomes. These results demonstrate that the cytoplasmic tail of CD1b determines the targeting of CD1b molecules to the limiting membrane of the lysosome.

Figure 3. CD1b tail targets the CD1b molecule to the limiting membrane of the lysosome. The localization of CD1b wt and CD1b tail chimera within LAMP1-positive compartments was determined in T2 cells transfected with the CD1b constructs (A). As a control, the localization of endogenous CD63 within these transfected cells was determined (B). Immunogold labeling with CD1b- or CD63-specific antibodies was performed on ultrathin cryosections, and the average number (and SE) of gold particles present on the limiting membrane and internal membranes was determined per lysosome.

Localization on the Internal Membranes of Lysosomes Does Not Determine Trafficking during DC Maturation

We next asked whether prior sublocalization in the internal or limiting membranes of lysosomes determines localization after DC maturation. If the internal membranes of the MIIC with MHC class II and its other molecules such as CD63 are all relocated after maturation, one would predict that CD63 would be absent from the MDL. However, immunogold localization of CD63 demonstrated its abundant presence on the electron-dense LAMP1-positive structures in MDC (Figure 4B). Thus, molecules present on the internal membranes of the MIIC can also be detected on the MDL. Also, CD1c molecules could be detected in these compartments but CD1a was not (Figures 2 and 5). Apparently, MHC class II molecules follow a different subcellular pathway in mature dendritic cells than CD1b, CD1c, CD63, or LAMP1. The segregation of the pathway might be at the level of the plasma membrane by constant internalization of CD1b, CD1c, CD63, and LAMP1.

Figure 5. Localization CD1c in immature and mature DCs. CD1c molecules are present in the multilamellar structures (A 10-nm gold) and in early endosomes of IMDCs (B 15-nm gold). Maturation of IMDCs to MDCs (C) shows the presence of CD1c molecules (15-nm gold, closed arrowheads) in MDLs (indicated by asterisks) and in early endosomes, whereas MHC class II molecules (10-nm gold, open arrowheads) are mostly present on the plasma membrane. Scale, 200 nm. p, plasma membrane G, Golgi complex e, endosomes and m, mitochondria.

CD1 Continues to Be Internalized from the Plasma Membrane in Clathrin-Coated Vesicles to a Similar Degree before and after Maturation

Besides localization to lysosomal MIICs, CD1b and CD1c were detected in early endocytic structures in IMDCs, suggesting internalization from the plasma membrane. However, previous studies on LPS-stimulated DCs have shown a sharp overall reduction of endocytic capacity after DC maturation (Inaba et al., 1993 Sallusto et al., 1995). Thus, we wished to determine whether clathrin-mediated endocytosis was blocked after maturation of DC. Internalization of transferrin conjugated with a fluorescent probe and internalization of CD1b detected with Fab-fragments against CD1b were used as markers for clathrin-mediated endocytosis in both MDC and IMDC. As a control, the fluid phase pinocytic capacity of MDC and IMDC was evaluated using fluorescent-labeled dextran. As expected, the uptake of the pinocytic marker dextran was reduced dramatically after DC maturation (Figure 6A). In contrast, the endocytosis of transferrin was not affected and remained at a constant level (Figure 6B). Also, we determined the localization of the endocytosed transferrin in the cells by using immunofluorescence and as expected, observed that the transferrin almost totally colocalized with EEA1 and transferrin receptor (TfR) (our unpublished data). Importantly, the plasma membrane levels of the Fab-fragments recognizing CD1b decreased in a similar manner during incubation at 37°C, in the absence or presence of LPS, suggesting equal internalization rates in MDC and IMDC (Figure 6C).

Figure 6. Endocytosis of dextran decreases but transferrin and CD1b internalization are unaltered after maturation. Receptor-mediated endocytic, pinocytic capacity and the internalization of CD1b were determined before and after stimulation with LPS (+LPS). Cells were incubated with fluorescently labeled dextran (A) or transferrin (B) and analyzed by flow cytometry directly after addition of the fluorescent probes (0-h incubation on ice) and after 1 h of uptake (1-h incubation at 37°C). Data represent the average fluorescent intensities measured in four different experiments, and the error bars indicate the SE. Maturation with LPS decreased pinocytosis nearly to half the value it was before maturation. The receptor-mediated endocytosis of transferrin was not affected by maturation. The uptake of transferrin used was below the saturation of the receptor as determined by titration experiments preventing fluid phase uptake. (C) The internalization rate of cell surface CD1b molecules labeled with Fab′ fragments on immature DCs (▪) and LPS-maturated DCs ( ). After internalization time of 30, 60, and 120 min, the average and the SE of relative mean fluorescence intensity are given. Monocyte-derived DCs of five different donors were used for five independent experiments. At t = 0, the labeling was not optimal most likely due to low temperature, and therefore the first measurement at 30 min is used to start calculating the graphs. The rate of internalization of the Fab-labeled CD1b molecules is not statistically different before and after maturation. (D) Percentages of early endosomes positive for CD1b or CD1c in IMDCs and MDCs. At least 40 different cells were used for detection of early endosomes that were classified as such using EEA1 labeling.

Next, using cryoimmunogold electron microscopy we determined the steady-state localization of CD1a, CD1b, and CD1c molecules to early endocytic compartments in IMDC and MDC. We noted that in contrast to the MIIC, no apparent change in the structure of the early endosomes was observed. CD1b and CD1c but not MHC class II were detected in structures morphologically similar to early endosomes, clathrin-coated pits, and coated vesicles in IMDCs (Sugita et al., 1996) and MDCs (Figure 5B). To ensure that these structures were early endosomes, colocalization studies with the TfR and EEA1 were performed. Both markers were detected in these CD1b-containing compartments. Early endosomes (immunogold labeled with antibodies against EEA1) in both unstimulated and LPS-stimulated DCs was counted, and the percentage in which colocalization with CD1b or CD1c occurred was determined. These percentages demonstrated that the amount of EEA-positive early endosomes in which CD1b or CD1c is present has not changed after maturation. Also, for CD1a localization, no quantifiable difference was noticed (our unpublished data). In IMDC and MDC, CD1a expression remained restricted to the plasma membrane and early endocytic tubulovesicular structures. Thus, we detected no differences in the occurrence of CD1 molecules in the early endocytic structures in mature compared with immature DCs, confirming the internalization data using fluorescent probes. Together, these studies emphasize that in contrast to MHC class II, CD1b (and c) molecules continue to be mainly in lysosomes, despite the drastic changes that occur in these structures. Such persistence in lysosomes, even after DC maturation, may be accounted for in part by the continued internalization from the cell surface.

No Up-Regulation of CD1 Cell Surface Expression after DC Maturation

Maturation of DCs has been shown to induce increased levels of MHC class II expression on the cell surface. Because the subcellular trafficking of CD1 molecules after maturation seems to differ dramatically from the MHC class II pathway, involving lysosomes and early endocytic structures, the effect of maturation on cell surface levels of CD1 was determined. IMDC were stimulated with LPS to develop into MDC. After LPS stimulation, virtually all the cells strikingly up-regulated the surface expression of CD83, one of the most reliable markers for DC maturation, confirming that all cells obtained in our experiments were MDCs (Figure 7). These cells also expressed markedly increased levels of MHC class II molecules on the cell surface. In contrast, up-regulation of cell surface expression was not readily detected for CD1 molecules. The surface expression of CD1a was apparently slightly down-regulated, whereas that of CD1b and CD1c was not changed after LPS stimulation. Similarly, the surface expression of LAMP1 and CD63 remained low and was not significantly altered. These data demonstrate that despite the fact that MHC class II, CD1b, CD1c, LAMP1, and CD63 were expressed in the same lysosomal compartments in IMDC, the up-regulation of surface expression after DC maturation was detected only for MHC class II, but not for other lysosomal residents. This finding further underscores how intracellular trafficking pathways of CD1b and CD1c differ from MHC class II.

Figure 7. LPS stimulation matures DCs but did not raise cell surface levels of CD1 molecules. Monocyte-derived DCs cultured in GM-CSF and IL-4 were analyzed by flow cytometry to determine the expression of cell surface markers. Similar analyses were done on cells stimulated by addition of LPS for 48 h and control cells kept in GM-CSF, IL-4 medium for that period. Maturation marker CD83 is like MHC class II up-regulated in LPS-treated cells. Cell surface levels are not raised for CD1a, CD1b, and CD1c and lysosomal markers CD63 and LAMP1 on the LPS-treated cells. Data shown are representative for six separate experiments in which monocytes from different donors were used.


Major Histocompatibility Complex Genomics and Human Disease

Over several decades, various forms of genomic analysis of the human major histocompatibility complex (MHC) have been extremely successful in picking up many disease associations. This is to be expected, as the MHC region is one of the most gene-dense and polymorphic stretches of human DNA. It also encodes proteins critical to immunity, including several controlling antigen processing and presentation. Single-nucleotide polymorphism genotyping and human leukocyte antigen (HLA) imputation now permit the screening of large sample sets, a technique further facilitated by high-throughput sequencing. These methods promise to yield more precise contributions of MHC variants to disease. However, interpretation of MHC-disease associations in terms of the functions of variants has been problematic. Most studies confirm the paramount importance of class I and class II molecules, which are key to resistance to infection. Infection is likely driving the extreme variation of these genes across the human population, but this has been difficult to demonstrate. In contrast, many associations with autoimmune conditions have been shown to be specific to certain class I and class II alleles. Interestingly, conditions other than infections and autoimmunity are also associated with the MHC, including some cancers and neuropathies. These associations could be indirect, owing, for example, to the infectious history of a particular individual and selective pressures operating at the population level.