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MHC A Review.

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1 MHC A Review

2 MHC Originally discovered in the 1930s by Peter Gorer (gorer, 1936)
MHC was genetically defined by George Snell (1948) Human Leukocyte Antigen (HLA) was sequences by Betck et al. in 1999 MHC is a complex multigene family comprising loci encoding receptors on the surface of a variety of immune and nonimmune cells These receptors bind amino acid fragments (or peptides) from foreign pathogens to start an immunological response

3 MHC The diversity of MHC genes underlies resistance to the diverisyt of infectious pathogens (Zuckerkandle and Pauling) “Classical” is used to indicate that an MHC gene is polymorphic and highly expressed “Nonclassical” genes are MHC genes that cluster with other MHC genes phylogenetically They are usually not expressed at high levels and are not polymorphic

4 General Organization and Inheritance of MHC
Every mammalian species studied possesses a tightly linked cluster of genes, the major histocompatibility complex (MHC) Associated with Intercellular recognition Self/nonself discrimination MHC plays a central role in the development of both humoral and cell-mediated immune responses

5 General Organization and Inheritance of MHC
Role in antigen recognition by T-cells Antigen-presenting structures TH and Tc cells Partly determines the response of an individual to antigens of infectious organisms and the MHC has been implicated in the susceptibility to disease and in the development in autoimmunity

6 Location and Function of MHC Regions
Chromosome 6 in humans = HLA The most gene-rich region, with over 200 loci identified Chromosome 17 in mice = MHC/ H-2 3 classes of molecules Class I MHC genes Encodes glycoproteins expressed on the surface of nearly all nucleated cells Present peptide antigens of altered self-cells necessary for the activation of Tc cells

7 Class I MHC genes Encodes glycoproteins expressed on the surface of nearly all nucleated cells Present peptide antigens of altered self-cells necessary for the activation of TC cells Encoded by the A, B and C regions in humans

8 Class II MHC genes Encodes glycoprteins expressed primarily on antigen-presenting cells Macrophages, dendritic cells and B cells Presents antigenic peptides to TH cells Encoded by DP, DQ and DR regions in humans

9 Class III MHC genes Encodes secreted proteins associated with the immune process including soluble serum proteins, components of the complement system and tumor necrosis factor C4, C2, Bf (complement proteins) in humans

10 MHC Individuals inherit the alleles as two sets, one from each parent.
Each set of alleles if referred to as a haplotype In an outbred population of humans the offspring are generally heterozygous at many loci and will express both maternal and paternal MHC alleles Co-dominantly expressed on the same cell

11 Generations If two inbred strains of mice having different MHC haplotypes are bred, the F1 generation inherits haplotypes from both parental strains and expresses both parental alleles at each MHC locus Congenic Mice = two strains that are genetically identical except at a single locus or region The first letter in a congenic strain refers to the strain providing the genetic background and the second letter to the strain providing the genetically different MHC region.

12 MHC Molecules and Genes
Class I and Class II MHC molecules are membrane-bound glycoproteins Antigen presenting molecules Class II MHC molecules are soluble proteins and exhibit much less polymorphism than class I and II

13 Structure of Class I Molecule
Contains a large α chain associated noncovalently with the much smaller β2-microglobulin molecule. The α chain is a polymorphic transmembrane glycoproteins 45 kilodaltons (kDa) Encoded by genes within the A, B and C region of the HLA in humans Anchored in the membrane by hydrophobic transmembrane segment and hydrophilic cytoplasmic tail β2-microglobulin is an invariant protein of about 12kDa encoded by a gene located on a different chromosome

14 Structure of Class I Molecule
α chain molecules are organized into three external domains (α1, α2, α3) each containing approximately 90 amino acids A transdomain of about 40 amino acids and a cytoplasmic anchor segment of 30 amino acids Β2-microglobulin is similar to α3 external domain Papain (enzyme) cleaves the α chain 13 residues proximal to its transmembrane domain

15 Structure of Class I Molecule
α1 and α2 domains interact for form a platform of eight antiparallel β strands spanned by two long α-helical regions This structure forms a deep groove or cleft approximately 25 Å X 10 Å X 11Å The long α helices as sides and the β strands of the β sheet as the bottom The Peptide Binding Cleft is located on the top surface of the class I MHC molecule Can bind 8-10 amino acids

16 Peptide Binding by MHC Molecules
Several hundred different allelic variants of class I MHC molecules in humans Each individual expresses only up to 6 different class I molecules and up to 12 different class II molecules A given MHC molecule can bind numerous different peptides Some peptides can bind to several different MHC molecules Class I molecules bind peptides containing 8 – 10 amino acid resides

17 Class I MHC-Peptide Interaction
Class I MHC molecules bind peptides Present these to CD8+T cells A nucleated cell expresses about 105 copies of each class I molecule Many different peptides will be expressed simultaneously on the surface of a nucleated cell by class I MHC molecules Class I MHC can bind over 2,000 distinct peptides Presented with a frequency of 100-4,000 copies per cell

18 Polymorphism of Class I Molecules
Polymorphism – Multiple alleles at a given genetic locus within a species MHC expressed by an individual does not change over time One of the most polymorphic genetic complexes Possesses an extraordinarily large number of different alleles at each locus The alleles differ in their DNA sequences from one individual to another The gene products expressed by different individuals have unique structural differences

19 Polymorphism of Class I Molecules
Up to 20 amino acid residues contributing to the uniqueness of each allele Analysis of HLA class I molecules has so far revealed 59 A alleles, 111 B alleles, and 37 C alleles. The current estimate of actual polymorphism in humans is on the order of 100 alleles for each locus The binding site for antigens is the most polymorphic location on MHC Class I

20 MHC Genes Chromosome 6 in humans and 17 in mice
Nearly 100 genes spanning some 4000kb 2000kb for Class I Contains approximately 20 genes HLA-A, HLA-B, HLA-C HLA-A has 250 and HLA-B has 500 known alleles Non-classical MHC class I include: HLA-E, HLA-F, HLA-G, HLA-H, HLA-J, HLA-X and MIC Many of these are pseudogenes and do not encode a protein product The non-classical gene products are categorized as class Ib molecules

21 Class Ib Molecules Have an α chain that associates with β2-microglobulin Less polymorphic Expressed at lower levels Tissue distribution is more limited than classical class I molecules The function of non-classical class I MHC molecules is largely unknown

22 Classical Class I MHC Molecules
Expressed on most somatic cells Level of expression varies Highest level of expression are by lymphocytes 1% of the total plasma-membrane proteins, 5X105 molecules per cell

23 Regulation of MHC Expression
Interferons (alpha, beta and gamma) and tumor necrosis factor increase expression of class I MHC molecules on cells MHC expression is increased or more commonly, decreased by a number of viruses, ie. Cytomegalovirus, hepatitis B virus and adenovirus 12.

24 Chemosensation and Genetic Individuality
Olfactory cues play a fundamental role is social interactions in a wide variety of mammals from rodents to primates (Brown, 1979) Each mammal has an individual odour or signature for individual recognition (Porter, 1985) Olfactory signatures have a function in mammalian reproduction (MacDonald and Brown, 1985) Chemical signals can be deposited on external substrates to mark territory Olfactory cues can still be effective in darkness (Porter, 1999)

25 Immunobiology of Classical MHC class I antigens
Outbreeding mammals have a degree of polymorphism that ensures that in each species there are more than 109 unique class I antigenic phenotypes in a population The heavy chain consists of three domains (α1, α2 and α3) The light chain consists of a single domain, β2-microglobulin, attached non-covalently to the α3 domain

26 Immunobiology of Classical MHC class I antigens
The α1 and α2 domains consist of an α-helix and a β-pleated sheet. The two sheets join together to make a continuous platform which supports the two α-helices defining an antigen binding cleft The cleft is occupied by a self-peptide of 8-10 amino acids

27 MHC-associated Odours and Individual Recognition
Inbred strains consist of animals that are genetically uniform and homozygous for all their genes Each chromosome pair is identical Resulting from mating brother and sister pairs for at least 20 consecutive generations The genotype is nearly identical Congenic strains are two strains that are identical except for allelic differences at a particular locus Any measurable characteristic that differs between the two congenic strains must be ascribed to the genes at that locus Formed by repeated backcrosses of a donor strain to an inbred strain with selection for a locus of interest

28 The Y-maze Paradigm Mice are trained to enter alternative chambers scented by an airflow through odour boxes occupied by MHC-congenic mice Mice are trained by water deprivation and reward The mouse first learns to differentiated between two strong odours like juniper and cinnamon and is rewarded with a drink It is correct if it chooses the correct arm at least 80% of the time The mice learn to distinguish inbred strains and then distinguish congenic strains that differ only in the alleles at the MHC

29 The Y-maze Paradigm Findings:
No sensory perception other than olfaction is required to distinguish between MHC types (Yamazaki, 1979) Urine is a good source of MHC-specific odours (Yamaguchi, 1981) Males and females had equal ability to distinguish between MHC types (Yamaguchi, 1981) Olfactory acuity of mice could enable the discrimination of MHC Class I molecules that differ only in three amino acids (Yamazaki, 1983a) No amount of training could enable a mouse to distinguish between individuals of the same inbred strain (Yamaguchi, 1981)

30 The Y-maze Paradigm Mice could discriminate between allelic differences in the class II and Q/T/M regions, indicating that all three genetic loci of the MHC can contribute to individual odours (Yamazaki, 1982, 1984) The haemopoietic system is a source of MHC-associated odours (Yamazaki, 1985) X and Y chromosomes are also discriminated between, but are much less potent in scent marking than the MHC (Yamazaki, 1986a) Ease of training indicates that MHC is a potent source of individual odour (Boyse, 1987)

31 Habituation-Dishabituation Paradigm
Most mammal are usually inquisitive when presented with a novel stimulus, but the novelty can wear off upon extended exposure to the stimulus The test involves placing an experimental animal in a test arena with a removable wire lid The animal is given opportunity to explore Then it is presented with the first urine odour on a piece of filter paper attached to the wire lid A novel stimulus

32 Habituation-Dishabituation Paradigm
The animal rears on its hind legs and sniffs The urine sample is presented three times By the 3rd exposure it no longer rears to investigate It has habituated to the odour A novel urine sample is presented to the animal If it perceives the odour of the new sample to be different from the first there is a rapid dishabituation The rat lifts its head, sniffs the air and moves toward the new odour and rears to investigate it

33 MHC-associated odours and reproduction
The male will choose a female that is different from him at the MHC (Yamazaki, 1976) Females can also choose mates and prefer to mate with males of different MHC (Potts, 1991) “Bruce Effect” – the pregnant female is introduced to a strange male during the preimplantation stage of development and spontaneous abortion occurs (Yamazaki, 1983b) The effect is likely due to a neuroendocrine disturbance

34 MHC-associated odours: the “carrier hypothesis”
MHC-based developmental variations give rise to distinctive odour profiles (Boyse, 1987) It is possible to discriminate between urine samples taken from germ-free MHC congenic mice, which lack commensal flora (Yamazaki, 1990) Fetal MHC-associated odours are evident in the urine of mothers as early as day 9 of gestation (Beauchamp, 1994) Before the fetus has a functioning immune system (Owen, 1969)

35 MHC-associated odours: the “carrier hypothesis”
A hypothetical route of class I MHC molecules from the membrane to the circulation and into the urine Cleavage of the molecule at the juxta-membranous protease site removes the transmembrane region and releases the molecule into the circulation in soluble form The molecule does not aggregate and circulates as a soluble heterodimer consisting of α and β chains Further enzymatic cleavage at the junction of the α2 and α3 domains removes most of the 3 domain with the attached β2-microglobulin This cleavage allows relaxation of the binding platform, opening of the cleft and loss of bound peptide

36 MHC-associated odours: the “carrier hypothesis”
Further enzymatic cleavage at the junction of the α2 and α3 domains removes most of the 3 domain with the attached β2-microglobulin This cleavage allows relaxation of the binding platform, opening of the cleft and loss of bound peptide The resulting molecule with a naked binding cleft is capable of binding a cocktail of small aromatic molecules such as volatile-odorants and is small enough to pass into the urine

37 MHC-associated odours: the “carrier hypothesis”
In the serum the molecule is a heterodimer with a heavy chain of 39kDa associated non-covalently with β2-microglobulin Present in the serum as a concentration between 350 – 390 ng/ml A half-life of 2.7 h Excreted via the kidneys In the urine degradation occurs giving rise to a major molecular mass species of 27kDa Cleavage at the papain cleavage site

38 MHC-associated odours: the “carrier hypothesis”
Class I molecules are large and consequently lack a vapour pressure Class I molecules might associate with smaller molecules, in an allelic-specific way, and transport them from the blood into the urine Serum has an odour but cannot be used to discriminate between MHC types (Brown, 1987, Yamazaki, 1999) Treat with proteases to liberate odour Carboxylic acids may be a source of the odour

39 In Search of the Chemical Basis for MHC Odourtypes (Kwak, 2010)
Lewis Thomas (1975) Two different classes of compounds have been proposed: Small volatile molecules and non-voltaile MHC peptide ligans (Boehm, 2005, Restrepo, 2006) Volatile molecules are non-ionic and have a finite vapour pressure between ambient and 250°C Their molecular weights are generally less than 300Da

40 Odourtype Volatile molecules are MHC peptide ligands are non-ionic and
have a finite vapour pressure between ambient and 250°C Their molecular weights are generally less than 300Da MHC peptide ligands are Ionic and Composed of inne amino acids Their molecular weights exceed 1000Da

41 Chemical Investigations of MHC Odourtype in Mice
Mice can recognize the MHC-driven odour cues from urine and blood Studies have failed to find specific behavioral effects of MHC variation in wild mice (Cheetham, 2007, Sherborne 2007, Thom 2008, Potts 1991)

42 Chemical Investigations of MHC Odourtype in Mice
Using anion exchange chromatography confirmed that acidic compounds were responsible for the MHC odour cues Some voltaile organic acids are regulated by MHC genetic variation (Singer, 1997) GC/MS study of about 80 compounds associated with MHC (Willse, 2005) MHC associated odour cue can be recognized in spite of variation in background strains (Beauchamp, 1990, Yamazaki, 1994, Eggert, 1996, Willse, 2006)

43 Chemical Investigations of MHC Odourtype in Mice
MHC-regulated metabolic odour signal must have a background-independent component GC/MS experiments on 3 MHC-congenic mice derived from different background strains 36 out of 55 peaks from urine were found to be present in all three strains Background genes, MHC genes and their interaction regulate the urinary volatile profiles SPME GC/MS on mice (Kwak 2009)

44 Chemical Investigations of MHC Odourtype in Mice
A compound was found to be more prominent in one MHC type in one strain and less prominent in that same MHC type in the other strain MHC regulation of volatile metabolic phenotypes was modulated by background genes All published chemical investigations have failed to find qualitative differences in volatile compounds that are associated with MHC types, except Eggert, 1996 The patterns of volatile metabolites vary according to MHC types, inconsistently and in a complex way

45 The Major Histocompatibility Complex and the Chemosensory Signaling of Individuality in Humans
Four characteristics of MHC is responsible for its enormous diversity A number of functional loci per class Allelopolymorphism (estimated for HLA class I raging up to 60 different alleles per locus) Codominant expression A high level of heterozygocity (in natural populations of mice, it is almost 100%)

46 The Major Histocompatibility Complex and the Chemosensory Signaling of Individuality in Humans
MHC effects on social behavior in humans came from three types of studies A series of population studies on the occurrence of shared HLA types in couples which in part confirms an influence of the MHC but are not conclusive Experimental studies on trained rats’ responses to human urinary odor which demonstrated preliminary results of an MHC-associated odor expression in humans A field study in which women were asked to index others whose body odors elicited a strong response A higher communality between indexing females and indexed females than with indexed males Males for whom negatively rated body odors were reported show a higher genetic similarity than those for whom positively rated body odors were reported (Eggert, 1994)

47 Investigate trained rats’ responses to human odors
Rats are able to distinguish between the urine odors of immunogenetically defined HLA-homogenous groups of persons HLA-associated urine donors occur in members of both sexes Stimulus generalization task can be performed using urine donors with different HLA-types HLA-associated odor constitution appears not to be limited to a definite HLA-specificity

48 Fractionation and Bioassay of Human Odor Types
Which fraction in human urine are chemosensorically active? The role of fractions containing either volatile or nonvolatile substances Bioassay using rats sniffing volatiles, smaller than 250Da and nonvolatiles Rats were able to recognize the odor profile of the training samples in the fractionated residue The distillate from the urine samples containing the volatile urine fraction was not recognized

49 GC of Human Urine Using a GC on human urine 120 peaks were found
16 out of 120 of specific components associated with the examined HLA-types were found HLA differences influence the pattern of ubiquitous metabolites Is the body odor of humans associated with HLA-types? The constitution of body odors in humans may provide evidence of an ultimate function of MHC-associated odors in humans

50 Fractionation and Bioassay of Human Odor Types
All tests showed that the bioactive information of the odor profile is not included in the distillate containing only the volatiles. Olfactory information was not changed by adding a foreign distillate to a familiar residue Olfactory recognition only occurred when the residue of the fractionated urine samples were presented Odor conveyed by the volatiles disappears very quickly. The mechanism of slowly released volatiles would prevent a rapid disappearance of the individual scent

51 MHC-regulated odortypes: Peptide-free urinary volatile signals
Small molecular weight compounds are involved in the signaling of MHC odortype It appears that MHC genetic variation influences both volatile and non-volatile (peptide-based) chemical signals It could be that volatile signals are used to recognize a member of the species and non-voltaile signals may play a more prominent role close up Humans may discriminate sweat odors of the subject with different HLA type and may have HLA-dependent odor preferences. This also occurs in mice

52 Olfactory Cues Associated with the MHC
Chemosenory signals are used by many species as markers of individuality helping to identify members of the same species A marker of individuality has to stable over longer times and must be very different than a genetic marker makes sense MHC also allows for degrees of genetic relatedness (kin recognition) MHC has to be phenotypically expressed in a way that allows other members of the species to evaluate the specific MHC-type present

53 MHC MHC codes for transmembrane glycoproteins
Self/non-self discrimination of the immune system MHC class I is also found in soluble form in the body fluids Serum, lymph and urine of rats In human A9;A23, A24 are expressed in large amounts in the soluble form Expression of odor signals is associated with the MHC and has been proposed that these mechanisms of natural selection have formed the development of a mechanism of sexual selection which is involved in the maintenance of the genetic diversity in the MHC

54 MHC Associated Odor Signals
Lewis Thomas was the first to propose that chemosensory signals associated with the MHC serve the function of discriminative stimuli How are MHC specific odors generated? Selective binding of odor molecules to soluble derivatives of MHC class I receptors or MHC specific selective bacterial colonizing or Coexpression of odor producing genes lying in the MHC region

55 MHC Associated Odor Signals
In humans There appears to be an association between the concentration of soluble MHC proteins and the salience of body odor cues Subjects with the HLA types A9 (A23, A24) and B15 (B62, B63) occur in a much higher proportion than expected in samples of subjects whose body odor was described as more salient than usual These subject also show an elevated concentration of soluble MHC proteins in their body fluids

56 MHC Associated Odor Signals
The concentration of soluble MHC proteins appears to be correlated with the reproductive cycle in women with the highest concentrations within the first half of the cycle The hedonics of the body odors of other subjects appears to be associated with the similarity with regard to the MHC

57 MHC Associated Odor Signals
In the same sex, body odors of similar subjects are rated more positive than body odors of dissimilar subjects In different sexes the reverse results were obtained The similarity of the HLA class I genotype of two person influences social perception measured by ratings of familiarity, attractiveness or pleasantness

58 MHC-correlated mate choice in humans
MHC in humans is also called Human Leukocyte Antigen (HLA)

59 Disassortative Preferences: A Theory
Women not using the contraceptive pill rated as more pleasant the odors of men with MHC antigens that were dissimilar to their own The smell of MHC dissimilar men also reminded them of their current or previous mate’s smell more frequently than did the smell of MHC similar men Women using the contraceptive pill preferred the smell of MHC similar men. Hormonal contraception physiologically mimics pregnancy and there is a shift in preference during pregnancy towards kin who would be most likely to assist in offspring care

60 Disassortative Preferences: A Theory
Women rated the odor of six T-shirts worn by four males and two females Both men and normally cycling women showed a tendency to rate the odors of MHC dissimilar subjects as more attractive through a negative correlation between the number of shared alleles and pleasantness ratings Pill users rated MHC similar odors as more pleasant

61 Disassortative Preferences: A Theory
T-shirt odors samples were collected from 6 men Female raters chose a sample which they would most and least like to smell on an everyday basis Most preferred odor donors who shared significantly more alleles (2.3 on average) A higher preference for the smell of donors carrying the same alleles as the smellers inherited from their fathers, but not their mothers (Hutterites)

62 Processing of MHC correlated odors
Brain activity was recorded in humans who were presented with two HLA similar and one HLA dissimilar stimuli MHC similar samples were processed faster and evoked larger potentials than MHC dissimilar samples Avoiding MHC similar partners (Inbreeding) may have higher biological significance that attraction to MHC dissimilar partners, whose odor elicited relatively weaker responses

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