While many factors can influence the fate of developing T cells
in the thymus, among the most influential are the strength and
timing of signals transmitted by the T cell antigen receptor
(TCR). Weak TCR binding to peptide/major histocompatibility
complex (MHC) ligands induces thymocytes to develop into naïve T
cells, a process called positive selection. Mechanisms that
prevent naive T cell development are termed “negative
selection” and include apoptotic deletion and agonist selection
into non-naive T cell lineages. Strong TCR binding to pMHC
ligands induces negative selection of thymocytes.
Thymocytes interact with two broad types of antigen-presenting
cells (APCs): bone marrow (BM)-derived APCs and thymic epithelial
cells (TECs). The traditional view of thymic selection is that
positive selection occurs in the thymic cortex via interaction
with TECs, followed by negative selection in the thymic medulla
via interaction with BM-APCs. However, recent studies reveal that
thymocytes can undergo either cortical or medullary negative
selection, which differ in terms of the phenotypes of the
thymocytes involved and their timing during T cell development.
It remains unclear which APC types are required to mediate these
two negative selection processes, which are called “Wave 1”
and “Wave 2” of negative selection. In addition, the
contributions of MHC class I (MHCI) and MHC class II (MHCII) to
the two waves are unclear.
To dissect thymic selection, two assays were used in this study.
First, the transcription factor, Helios, was used in a flow
cytometry assay as a marker of negatively selected cells, in
conjunction with chemokine receptor-7 (CCR7) to distinguish
thymocyte maturation stages. Apoptosis-defective mice were used
to inhibit death of negatively selected cells, allowing direct
quantification of negative selection. Genetic ablation of MHCII
expression within BM-APCs or autoimmune regulator (Aire–/–)
within TECs was used to examine the roles of APC types in thymic
selection. Mice lacking expression of MHCI and/or MHCII were also
examined. Second, a TCR sequencing assay was used to examine the
characteristics of positively and negatively selected TCR
repertoires in mice lacking expression of MHCI and/or MHCII.
In TCR transgenic and polyclonal models, MHCII+ BM-APCs were
required to induce wave 1 negative selection. Ablation of MHCII+
BM-APCs either abrogated negative selection completely or delayed
negative selection from wave 1 to wave 2. Although MHCI and MHCII
were found to induce similar frequencies of TCR-signalled
thymocytes, MHCII was found to make a greater contribution to
negative selection than MHCI. Sequencing data revealed that TCRs
that provoke negative selection at wave 1 are enriched with
hydrophobic amino acids in the region expected to interact with
the peptide component of pMHC ligands. Interestingly, hydrophobic
amino acids are also enriched in the same region of TCRs that
transmit a signal in mice lacking MHC expression. These results
provide new insight into the determinants of thymic negative
selection
