Author's School

Graduate School of Arts & Sciences

Author's Department/Program

Biology and Biomedical Sciences: Immunology

Language

English (en)

Date of Award

Summer 9-1-2014

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Chair and Committee

Paul M Allen

Abstract

Critical in determining transplantation outcome, whether tolerance is achieved or not, are CD4+ T cells that can recognize peptides presented on allogeneic (non-self) Major Histocompatibility Complex (MHC) molecules in addition to their conventional ligands, peptides presented on self MHC. It is an enigma as to how these alloreactive T cells can bind to allogeneic MHC given that T cells undergo stringent positive selection during development to bind to peptides presented on self MHC. We hypothesize that T cells bind to peptides on non-self MHC using the same properties involved in binding their conventional ligands. We identified allostimulatory peptide-MHC (pMHC) ligands (I-Ek as the allogeneic MHC) for two LLO/I-Ab-specific CD4+ T cells, LLO118 and 1G5.1. Both T cells recognized their allostimulatory ligand with a high degree of specificity and sensitivity for the allopeptide, similar to how they recognized their cognate ligand. Allopeptide recognition was also shown to not merely reflect mimicry of the cognate peptide. The integral role of the peptide in alloreactivity was further confirmed by the ability to convert previously non-alloreactive T cell hybrids into becoming responsive with the addition of peptide pools. In addition, the binding affinity and kinetics of LLO118 to its allostimulatory and cognate pMHC ligands were compared using surface plasmon resonance (Biacore system). LLO118 bound its alloligand using similar affinity and kinetics compared to its cognate ligand. In comparing alloreactivity with conventional recognition, which has not been done before for CD4+ T cells, we have found that similar peptide specificity and binding affinity are used to recognize both ligands, shedding light on the fundamental binding properties of the T cell receptor (TCR) for pMHC.

Within the population of alloreactive T cells, a significant percentage is comprised of dual TCR T cells. This occurs from incomplete allelic exclusion of the TCRα loci during thymic development, allowing for simultaneous rearrangement of TCRα on both alleles until positively selecting signals are received through the TCR. In dual TCR T cells, only one TCR needs to mediate positive selection, and an autoreactive TCR can be masked from negative selection through decreased surface expression. This generates a repertoire of T cells containing secondary TCRs unconstrained by thymic selection. We set out to investigate the impact of secondary TCRα rearrangement to determine what benefit this has on thymic development and further define its contribution to peripheral T cell responses. Our hypothesis is that secondary TCRα rearrangement positively impacts the development of T cells, but atypical TCR properties that arise contribute to alloreactivity and autoimmunity. We examined mice heterozygous for the T cell receptor α chain constant region (TCRα+/-), which have only one functional TCRα rearrangement. The mice had a defect in generating mature T cells attributable to decreased positive selection. Elimination of secondary TCRs did not broadly alter the peripheral T cell compartment, though deep sequencing of the TCRα repertoire demonstrated unique TCRs resulting from secondary rearrangements. The functional consequence of these unique TCRs was evidenced by the significantly reduced frequencies of TCRα+/- T cell binding to autoantigen and alloantigen pMHC tetramers as well as decreased in vivo alloreactivity. Analysis of responses to altered peptide ligands (APLs) revealed that dual TCR T cells had increased flexibility in their recognition of allogeneic ligands, indicating a mechanism for their importance in alloreactivity. Our results show that the role of secondary TCRs in alloreactivity appears to be more significant than what has been assumed.

Another factor we wanted to investigate in alloreactivity - as well as in autoimmunity and conventional T cell responses - is the effect of MHC composition, specifically the impact of increasing the types of MHC molecules expressed. It is perplexing why vertebrates express a limited number of MHC molecules when theoretically, having a greater repertoire of MHC molecules would increase the number of epitopes presented, thereby enhancing thymic selection and T cell response to pathogens. It is possible that any positive effects would either be neutralized or outweighed by negative selection restricting the T cell repertoire. We hypothesize that the limit on MHC number is due to negative consequences arising from expressing additional MHC. We compared T cell responses between B6 mice (I-A+) and B6.E+ mice (I-A+, I-E+), the latter expressing a second class II MHC molecule, I-Eb. The naive TCR Vβ repertoire was altered in B6.E+ thymi and spleens, suggesting a potential for mediating different outcomes in T cell reactivity. In alloreactivity, the B6.E+ T cell response was significantly dampened. We wondered if similar effects would be seen in other types of immune responses. The B6 and B6.E+ responses to hen egg-white lysozyme (HEL) protein immunization remained similar, but the quality of the T cell response was subtly altered in viral infection and there was markedly enhanced susceptibility to experimental autoimmune encephalomyelitis (EAE) in B6.E+ mice. The EAE phenotype could be explained by decreased percentage of natural regulatory T cells (nTregs) in the B6.E+ mice. Our data suggest that expressing an additional class II MHC can produce both positive and negative effects on a wide range of T cell responses. In conclusion, new insight into CD4+ T cell alloreactivity has been gained, with our research indicating that specificity of peptide binding, weak affinity, flexibility in recognition by dual TCR T cells, and MHC composition all contribute significantly to allorecognition.

DOI

https://doi.org/10.7936/K7QJ7FBS

Comments

This work is not available online per the author’s request. For access information, please contact digital@wumail.wustl.edu or visit http://digital.wustl.edu/publish/etd-search.html.

Permanent URL: http://dx.doi.org/10.7936/K7QJ7FBS

Share

COinS