© 1999 Robert A. Freitas Jr. All Rights Reserved.

Robert A. Freitas Jr., Nanomedicine, Volume I: Basic Capabilities, Landes Bioscience, Georgetown, TX, 1999

8.5.2.1 Identification of Self

The cytochemical distinction between self and nonself is mediated by the histocompatibility molecules.^{939,953-955,997} These antigens are genetically coded in the major histocompatibility complex (MHC), a cluster of ~28 genes located on chromosome 6 and comprising ~0.1% of the entire human genome. There are three broad classes (I, II, and III) of MHC genes that encode self antigens. The major function of the MHC-I and MHC-II molecules⁴³⁹ is the presentation of antigenic peptide fragments, derived from internal (MHC-I) or external (MHC-II) foreign proteins, to the immune system. It is the combination of an MHC molecule plus a foreign peptide fragment that appears antigenic to the immune system. (See Section 15.2.3.1.2.)

Most important are the MHC Class I molecules -- glycoproteins encoded by several separate genetic loci which comprise the HLA (histocompatibility locus antigens, formerly "human leukocyte antigens") complex. The three classical loci within the HLA complex are called HLA-A, HLA-B, and HLA-C. Essentially all adult nucleated cells in the human body (including white cells but excluding cells in the nervous system and cells of some tumors) display the classical MHC molecules of the A, B, and C regions on their surfaces in varying amounts.* Class I glycoproteins account for up to 1% of the total protein of the plasma membrane.⁹³⁹ Figure 8.33 shows the structure and orientation of a Class I MHC glycoprotein. Each MHC Class I molecule has two parts. The first part is a long folded glycosylated polypeptide chain of molecular weight ~45,000 daltons (~340 residues). This chain has a short (~30 residue) hydrophilic tail inside the cell and a ~40 residue hydrophobic transmembrane segment. The extracellular portion has three ~90-residue segments designated a₁, a₂, and a₃ with intrasegment disulfide bridges. Alloantigenic sites (carrying determinants specific to each individual) are located primarily in the a₁ domain and to a lesser extent in the a₂ domain, and there is a carbohydrate unit attached to the a₂ domain. The a₃ domain is relatively invariant. The second part of the MHC Class I molecule is a nonglycosylated 96-residue peptide called b₂-microglobulin, of molecular weight ~12,000 daltons, which is noncovalently bound to the a₃ domain of the longer glycosylated chain nearest the outer surface of the cell membrane. b₂-microglobulin is not part of the active antigenic site of the HLA molecule but is essential for the expression of specificity.

* Classical HLA tissue typing relied solely upon observations of cytotoxic reactions of human alloantisera with live lymphocytes. By 1998 it was apparent that this approach fails to distinguish all HLA alleles, and typing based on nucleotide sequences had begun to replace serological methods.¹⁰²⁸

MHC Class II molecules are also glycosylated integral membrane proteins (Fig. 8.34). These proteins are coded by at least six expressed genes located in the D region of the MHC cluster, each of which encodes either a ~34,000-dalton a chain or a ~28,000-dalton b chain. Each MHC Class II polypeptide consists of two external 90-residue domains, a 30-residue transmembrane domain, and a cytoplasmic domain with 10-15 amino acids. There are four serologically-determined Class II subtypes: HLA-DR and HLA-D (one a and several b chains), HLA-DQ (one a and one b chain), and HLA-DP (at least one a and one b chain). Each Class II glycoprotein molecule consists of at least two of these transmembrane polypeptide chains bound together noncovalently. Only the shorter b chains contain the alloantigenic sites, and both chains have carbohydrate units.⁹⁵⁵ MHC Class II molecules are less widely distributed than those of Class I, being found primarily on dendritic cells, glial cells in the brain, some epithelial and endothelial cells (e.g., Langerhans cells in the epidermis), monocytes, macrophages (including tissue macrophages such as the Kupffer cells of the liver), melanoma cells, activated (but not resting) T cells, and most B lymphocytes.^953,955 Other cells may be induced to express MHC Class II proteins when exposed to g interferon.⁹⁵⁶ Most parenchymal cells do not express MHC Class II molecules.⁹⁵³

MHC Class III genes encode the ~20 proteins that comprise the complement system, including Bf, C2, C4a and C4b. These are functionally related in that they are each involved in the activation of the C3 component (Section 15.2.3.2). They allow human cell membranes to be distinguished from nonhuman (e.g., bacterial) membranes, a crude form of self identification, but are nonspecific and thus play no role in distinguishing the cells of one patient from those of another.

The >3.8 megabase gene locus of the MHC is the most polymorphic (having alternative types) in the human genome; the number of observed MHC (HLA) types appears to be near the optimum number based on evolutionary simulation experiments using a cellular automaton model of antigen-lymphocyte interactions.⁹⁵⁸ Table 8.13 summarizes classical serological and modern nucleotide-based HLA specificities that were recognized as of 1998. (Not shown are several "public antigens" such as HLA-DRw52 and -DRw53 which, like MHC Class III proteins, are essentially nonspecific.) The HLA-uniqueness of an individual patient can only be estimated because the specificities do not occur with equal probability. For example, 16% of the human population has HLA-A1 but only 10% have HLA-B8.⁹⁵⁵ As an additional complication, the specificities are not strictly independent -- given the above probabilities, the combination A1B8 should occur with a frequency of 1.6%, but the actual observed frequency is 8.8%.⁹⁵⁵ Members of the same geneological family are also far more likely to have matching MHC Class II proteins than randomly selected unrelated individuals. Subject to these provisos, the existence of ~500 million distinct classical HLA combinations originally seemed to imply that only ~10 people in a worldwide population of 5 billion people shared exactly the same self-molecules, making each person literally "one in a billion." However, based on nucleotide sequencing, as of 1998 there were estimated to be >10¹⁶ known combinations, making each person quite histocompatibility-unique.

MHC Class I and Class II self-molecules are manufactured in the rough endoplasmic reticulum (Section 8.5.3.5) and are then delivered to the plasma membrane (Section 8.5.3.2) for presentation to the extracellular environment. A typical cell may have 15,000-30,000 Class I molecules (~10-20/micron²) at its surface; a cell expressing Class II proteins may display 50,000-500,000 Class II molecules (~40-400/micron²) at its surface.^439,3453 A nanorobot chemotactic sensor pad (with appropriate reversible binding sites; Section 4.2.8) measuring 50-300 nm in diameter pressed against a cell surface will overlay at least one of each class of self-molecule. Vesicle-transported proteins (like MHC proteins) have an estimated lateral diffusion coefficient in the cell plasma membrane of ~2 x 10^-14 m²/sec,⁵³¹ so an MHC molecule (assumed diameter ~10 nm) experiences an effective absolute viscosity of h_antigen ~ 2.3 kg/m-sec (Eqn. 3.5) and thus migrates across the plasma membrane surface under the sensor pad from center to periphery of the sensor pad in 20-600 millisec (vs. 0.01-0.5 millisec for phospholipid molecules in the membrane; Eqn. 3.1), allowing a single binding event and hence a detection in much less than a second.

Consider a chemotactic sensor pad of area A_pad overlaying N_antigen antigen molecules, comprising an array of N_sensor chemosensors each of area A_sensor. If a single detection event requires t = 3 p h_antigen R_antigen DX² / kT seconds (Eqn. 3.1) where DX is the mean antigen migration distance in the cell membrane, then the time needed to measure N_spec specificities assuming a minimum of N_encounter contact events to ensure a binding event is t_meas = t (N_spec N_encounter / N_sensor). Since N_sensor = A_pad / A_sensor and DX² = A_sensor N_spec / N_antigen, then:

{Eqn. 8.5}

Taking R_antigen = 5 nm, N_encounter = 10, A_sensor = 100 nm², A_pad = 90,000 nm², N_antigen = 1, and T = 310 K, then t_meas ~ (3 x 10^-5) N_spec². A nanorobot searching for a particular set of 7 HLA proteins (N_spec = 7) thus requires t_meas ~ 0.001 sec to make the self/nonself determination for a particular cell membrane it has encountered. A nanorobot seeking to determine the HLA type of the membrane (e.g., in mapping mode) must in the worst case search all (266 + 314 =) 580 HLA protein types (N_spec = 580), requiring at most t_meas ~ 10 sec.

The principal exceptions to the MHC system are the red blood cells, the most numerous native cells in the human body (Section 8.5.1). Red cells do not express HLA proteins. Rather, the erythrocyte surface expresses a complex set of at least 22 blood group systems and 7 antigen collections, as listed in Table 8.14a / 8.14b, plus 47 additional high-prevalence (public) and low-prevalence (private) antigens not associated with known systems or collections, also grouped into numbered series (not shown in the table).⁹⁵⁷ Some of these antigen systems are carbohydrates, like the familiar ABO system, which is coded by genes located on chromosome 9; others are nonglycosylated proteins, glycoproteins or glycosphingolipids. Each system or collection is expressed on every erythrocyte surface, so an exhaustive assay of all 30+ antigen systems allows at least ~10¹⁹ different combinations to be distinguished. Most combinations are extremely rare, so the practical net specificity of the entire blood group set is considerably less in the human population, though probably still much higher than the HLA system.

ABO is the best-known blood group system. Erythrocytes are typed as A, B, AB, or O, the latter indicating a lack of expression of either A or B. The H antigen is the precursor of A and B (Fig. 8.35) and is found on all red cell surfaces (up to ~1.7 x 10⁶ antigens/RBC, or ~18,000/micron²) except those of patients with the rare O_h Bombay or H-null phenotype. Because H is a precursor of A and B, type O erythrocytes have more H antigen than A or B erythrocytes, which in turn have more H antigen than AB erythrocytes (which express both A and B antigens). The number of A and B antigens on the red cell surface ranges from 12 x 10⁶ (~10,000-20,000/micron²); in 75% of Type A individuals, "double-length" A antigens are also present (~500/micron²). MNSs factor antigens range from ~2700-5400/micron², Rh factor antigens ~100-300/micron², Lewis factor antigens ~30/micron², and so forth. Again assuming a ~(300 nm)² chemotactic sensor pad, from Eqn. 8.5 a nanorobot searching for a particular set of ~30 blood group antigens (N_spec = 30) requires t_meas ~ 0.03 sec to make the self/nonself determination for a particular red cell membrane it has encountered. A nanorobot seeking to determine the complete blood group type of the membrane (e.g., in mapping mode) must in the worst case search all 254 known blood antigen types (N_spec = 254), requiring at most t_meas ~ 2 sec.

Direct detection of blood group antigens, or of antibodies to blood group antigens in body fluids, permits at least partial self-recognition by bloodborne nanorobots without the need for any direct cell contact, which may be useful in establishing theater protocols (Chapter 12). ABH, Lewis, I and P blood group antigens are found in blood plasma, and serum IgM-class antibodies associated with the carbohydrate antigens of the ABO, Lewis, and P blood group systems are almost universal. Anti-M and anti-N are common, anti-Sd^a is found in 12% of normal people, and anti-V^w or anti-Wr^a is found in ~1% of patients.⁹⁶⁰ In persons who previously have been pregnant or transfused, 0.16-0.56% have anti-D (Rh group) and 0.14-0.60% have anti-E and anti-C (Rh system), anti-K and anti-Fy^a, and several other antibodies in serum.⁹⁶⁰ Body secretions contain ABH, I and Lewis antigen but no P system antigens; Sd^a antigen is found in most body secretions, with the greatest concentration in the urine.⁹⁶⁰

Other partial exceptions to the MHC system are the keratinocytes, smooth muscle cells, and fibroblasts, which do not constitutively express HLA Class II molecules.⁹⁶⁷ As a result, foreign fibroblasts cannot induce the generation of required helper T cells and thus stimulate no rejection response when transplanted between hosts.

Last updated on 16 April 2004