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Parasites and pathogens impose selection pressures on their hosts that are not only strong but
also constantly changing as parasites adapt to host defenses or new pathogen strains become
abundant because of environmental fluctuations. Parasite mediated selection is believed to be
a major selective force responsible for the evolution of sexual reproduction, the maintenance
of genetic variation and the evolution of mate choice. It can drive local adaptation and
speciation. Since selection exerted by parasites is implicated in so many key topics in
evolutionary biology, it is of central interest to understand how defense mechanisms evolve.
Major histocompatibility complex (MHC) molecules play a central role in the adaptive
immune response of vertebrates. They present pathogenic peptides on the cell surface where
they can be recognized by T-cells with their T-cell receptor (TCR). This leads to the initiation
of an immune response. Every MHC molecule can present only peptides that match its
peptide-binding groove, so that a single MHC molecule confers resistance to some but not all
pathogens. Numerous studies have documented associations between resistance and the
presence of specific MHC alleles. However, the exact causes and mechanisms of pathogen
mediated selection on the MHC are only partly resolved. For example, it is unclear why
individuals that express an intermediate number of different MHC molecules are often more
immunocompetent than individuals that express many different MHC molecules and why
susceptibility MHC alleles are often not recessive but codominant when they occur in
combination with resistance alleles.
While existing studies on host-pathogen coevolution typically characterize the adaptive
immune system only by MHC genotyping host individuals, this thesis is dedicated to extend
the focus to T-cells and their TCRs. Our model system is the threespined stickleback
(Gasterosteus aculeatus). There are two main incentives to study T-cells and T-cell diversity
in this model species for evolutionary ecology. On the one hand research on T-cells may
directly help to unravel selection pressures operating on the MHC. On the other hand
comparative immunology, that is the study of the similarities and differences between immune
systems, can help to enhance our understanding of immune system function and evolution.
In the first chapter (my co-authors and) I model the effect of an increase in intra-individual
MHC diversity on T-cell repertoire diversity. Since MHC molecules present both self and
pathogenic peptides, T-cells have to be screened for self-tolerance during their maturation in
order to prevent autoimmune reactions. Self-reactive T-cells are eliminated in this process
termed negative selection. As intra-individual MHC-diversity increases, more and more selfpeptides
are presented, so that T-cell loss during negative selection increases. Individuals of
high MHC-diversity may have reduced immunocompetence because of T-cell repertoire
depletion: Their MHC molecules can present many pathogenic peptides, but they may lack Tcells
with the appropriate TCR to recognize them. The presented model suggests that this can
explain why individuals of intermediate intra-individual MHC diversity are most resistant.
The model can be tested by quantifying the loss of TCR specificities during negative selection
in individuals of low and high MHC diversity. To this end I characterize the TCRß loci and
the thymus of stickleback in chapters two and three and discuss how TCRß diversity can be
assessed in chapter four.
The loci encoding the ß chain of the TCR do not contain functional genes in the germline
state. Instead they consist of arrays of subgenic fragments that are rearranged in an essentially
random process during the maturation of each individual T-cell. Based on the published
reference genome, BAC-sequencing and linkage analysis I show that sticklebacks – in
contrast to all other species studied so far – have two unlinked TCRß loci, which rearrange
independently during T-cell maturation and are both expressed in mature T-cells. The
extraordinary genomic organization likely influences TCRß repertoire diversity and offers a
unique opportunity to gain insights on how allelic exclusion is enforced. I discuss the
phylogeny of the subgenic fragments at the two loci that partly arose by block duplication. An
interesting allelic variant is a six amino acid repeat motif in the cd loop of the conserved Cß
fragment. A complete list of all subgenic fragments allows assessment of the potential TCRß
repertoire, defined as the set of all TCRß sequences an individual can produce. This is a
robust basis for PCR-based approaches that aim at characterizing realized TCRß repertoires.
The thymus provides the microenvironment in which T-cells rearrange their TCR loci and are
selected for self-tolerance. In stickleback it is a paired organ in the dorsal region of the gill
cavity. Based on the distribution of stromal and parenchymal cell types as well as the
expression of recombination activating gene-1 and MHC, two zones can be differentiated that
resemble the mammalian cortex and medulla morphologically and functionally. Early
developmental stages of T-cells are located in the cortex. Negative selection takes place upon
entering the medulla. The anatomical separation of T-cells prior to and post the negative
selection phase, opens up the opportunity to study the diversity loss during selection by
comparing TCR diversity in the two thymic zones.
With a death rate greater than 95% among maturing T-cells, the thymus hosts a developmental
process in which the yield of functional cells is less than that of any other organ system. When
resources are allocated to this process differs significantly between species. I show that the
stickleback thymus develops early in ontogeny. Completely rearranged TCRß genes could
already be detected one week post fertilization. Two clearly demarcated zones are present in
the thymus from four weeks post hatching. This suggests that sticklebacks may be able to
mount an adaptive immune response one or two months post-hatch.
In ongoing work I seek to describe the composition and diversity of realized TCRß repertoires
in stickleback. Based on the description of TCRß gene segments in chapter two, primers were
designed that amplify the most diverse region of rearranged TCRß genes. This region encodes
for the complementarity determining region 3 which is in direct contact with the peptide when
the TCR interacts with a peptide-MHC complex. Extensive cloning and sequencing of
amplicons reveals that the repertoire of naive sticklebacks is diverse but contains a higher
proportion of non-functional rearrangements and fewer insertions than the human repertoire.
This work provides a basis for exhaustive sequencing and comparison of TCRß repertoires
e.g. in pre- and postselection T-cell pools or naive and infected individuals.
Based on the knowledge of the selective forces acting on a trait, mathematical models can
explore how it will spread in a population. In the fifth chapter I explore models that describe
the spreading of traits under frequency-dependent selection. In models of evolutionary game
theory frequency dependent selection is implemented by assigning fitness to individuals
according to a payoff obtained in an evolutionary game. Traditional evolutionary game theory
has derived deterministic differential equations that describe the spreading of traits in
infinitely large populations. However, in populations of finite size noise plays a major role in
evolutionary dynamics. I analyze two scenarios in a finite population. In the first scenario
individuals obtain a payoff based on interactions with a representative sample of the
population, so that all individuals of the same phenotype obtain the same payoff. In the second
scenario payoff evaluation is based on a single interaction with a randomly drawn individual,
so that individuals of the same type can have different payoffs. While the evolutionary
dynamics of the two scenarios are identical under weak selection, the evolutionary dynamics
become very different under strong selection
