CONTROL-T RESEARCH PROJECT 3
Dr. Jörg Kirberg
Dr. Sospeter Njeru
Second Funding Period
Accomplishments of the first funding period
In the first funding period one focus of RP3 was to delineate how competition among peripheral T cells is limited in order to preserve the normal polyclonal nature of the T-cell repertoire. The question arose since previous work of RP3 had shown that T cells expressing different TCRs could compete even when they would not share the restriction element. The results of RP3 now demonstrate that at least for some TCRs, the local availability of the homeostatic proliferation triggering self-peptide/MHC is not uniform, such that homeostatic proliferation occurs initially/mostly at specific sites. In this way, a variation component is added, changing the hierarchical position of the given T cells in relation to their competitors. In other words, interclonal competition is stratified by location. It is likely that this is because the relevant peptide ligand(s) is/are derived from tissue-specific self-antigens. Indeed, even under germ-free condition, the activating stimulus for one TCR was shown to be localized, demonstrating its endogenous origin. Central to T-cell homeostasis would then become T-cell recirculation. Indeed, experimentally offsetting the migration into lymph nodes by blocking CD62L showed that T cells are relatively residential when collecting the homeostatic-proliferation triggering signals - and this occurred at specific sites only. Together with RP4 the dynamics of T-cell recirculation were modeled to exclude other interpretations. While the aforementioned work used TCR-transgenic cells, recent results indicate that this phenomenon is universal, detectable even in a setting of a polyclonal TCRα chain repertoire. Thus, tissue-specific local antigens offset T-cell hierarchies to maintain clonal diversity. Underway are now studies together with RP5 on the alteration of the relative hierarchical position by MTCL-common oncogenes.
Mature lymphocytes persist over long periods of time while recirculating through the peripheral lymphoid organs. For T-cells, regular contact of the T-cell receptor (TCR) to restricting self-MHC molecules is essential for survival and may trigger homeostatic proliferation upon lymphopenia. Since TCRs vary with respect to affinities for their MHC-ligand and since T-cell homeostasis is also ruled by competition in trans, it is so far unclear
how a broad TCR repertoire is actually maintained. In the first funding period we could show that different TCRs can escape interclonal competition since the relevant self-peptide/MHC ligand essential for homeostatic proliferation is stratified by location, probably because the peptide ligand is derived from tissue-specific self-antigens. These results shall be extended using non-TCR transgenic model systems to demonstrate the wider applicability of these findings. We thus aim to clarify the processes behind the homeostasis of T-cells, apparently balancing the competition among T-cells bearing TCRs of varying affinity. Furthermore, the impact of these processes in the development of mature T-cell lymphoma (MTCL) will be addressed, using oncogene transgenic mice (collaboration with RP5) and/or retroviral transduction (RP2). Our findings appears relevant for T-cell lymphoma since previous work by CONTROL-T coworkers (RP2) showed that the time to MTCL outgrowth was much shorter in an oligoclonal TCR situation, implicating homeostatic processes in the pathogenesis of this disease. Elucidating these processes will improve our understanding of T-cell population dynamics and may give additional handles to improve MTCL treatment.
Aim A1: The role of newly identified genes from murine and human MTCL on T-cell homeostasis
MTCL candidate genes (RP1,2,5,7) expressed in monoclonal mature T-cells are tested in competitive reconstitution assays. Determine if changes in homeostatic potential result.
Aim A2: The role of TCL1 as a TCR proximal modulator in setting peripheral T-cell homeostasis / competitiveness in vivo
TCL1 modulates TCR signaling (RP5). Check impact of TCL1 on selection in P14-TCR-tg system. Perform reciprocal adoptive transfers (Ly-5 allele marked).
Aim B1: Altered T-cell hierarchy by specific self-peptides: Origin, identity, and presenting cell population
Germ-free OT2-TCR Rag-/- mice (CD69), blocking class II in vivo, APC ablation.
Aim C: The role of localized self-peptides and lymphocyte recirculation for the maintenance of a diverse TCR-repertoire
1-TCRα mice: Altered self-peptides vs. TCR-repertoire, NGS for analysis.
First Funding Period
Given their relatively constant number, peripheral T cells appear to be under control by homeostatic mechanisms. It has been established that the interaction of the T cell receptor (TCR) with self-MHC molecules determines a T cell’s potential to compete for, apparently, soluble factors. In this way, some T cells can limit the homeostatic expansion of other T cells, even in trans (Kirberg et al. 1997, Agenès et al., 2008, Takada and Jameson, 2009). A hypothesis to be tested within the CONTROL-T consortium is whether pathologic conditions such as T cell lymphoma may result because pre-leukemic cells overrule these normal mechanisms of homeostatic competition. In this way, tumor cell outgrowth could originate from cells that attained an increased homeostatic fitness relative to the pool of T cells. For this reason, but also generally, we aim to identify pathways specifically ruling T cell interclonal competitiveness. To this end, we will introduce genetic alterations into various homeostatic model systems to elucidate their role in this process. This will combine candidate genes, e.g. as identified by CONTROL-T partners, but also insertional mutagenesis-screens to specifically identify pathways altering T cell competitiveness.
Our work follows two lines of investigation:
Aim 1: Understanding cellular mechanisms of T cell homeostasis
T cell hierarchy and the role of endogenous antigens; consequences of (partial) agonist expression on T cell hierarchy and autoimmunity.
Aim 2: Factors (genes) relevant for homeostatic proliferation and implicated in T cell lymphoma outgrowth
Two lines of investigations shall be followed, congruent with the major aim of the CONTROL-T consortium, which is (I) to understand mechanisms of peripheral T cell homeostasis and (II) to relate those findings to the emergence of T cell lymphomas as a possible result of nonfunctional/defective homeostatic competition/control. The first aim is thus to investigate which influence self-antigens have on T cell competition/homeostasis. Specifically we will elucidate whether the diversity of self-peptides could mechanistically contribute to homeostasis and lymphocyte competition. The rules of inter-clonal T cell competitiveness will thus get addressed. In this way we hope to resolve the dichotomy regarding peripheral T cell competition when comparing CD4+ and CD8+ T cells (Agenès et al., 2008; Moses et al., 2003). The second major aim is to study processes that allow T cells to escape homeostatic competition. Here cells may acquire an increased homeostatic fitness by certain mutational/epigentic alterations, such that early pre-malignant T cells escape inter-clonal competition. This would lead to their gradual and, eventually, to their full transformation when further acquiring major transformation driving mutations. Previously, an in vivo retroviral mutagenesis screen had been performed in this lab in order to identify genes that overcome the developmental block in thymic positive selection when the restricting MHC molecule complexes were absent specifically on thymic epithelial cells. In this situation, the block in T cell development had to be overcome by mutagenesis events. Following such, T cell expansion by homeostatic proliferation took place, allowing for the analysis of sizeable T cell numbers using cell sorting followed by splinkerette/ligation-mediated PCR to identify proviral integration sites (methodology as in Mikkers et al., 2002, Kirberg et al., 2005, Newrzela et al., 2011). Obviously, in the screen the expansion of peripheral T cells occurs by homeostatic proliferation, along ongoing mutagenesis. Thus, some of the identified integration sites derived from this screen may have occurred late, promoting T cell outgrowth, and thus potentially have a role in T cell homeostasis. This is in line with the work of our collaboration partners in the CONTROL-T consortium, demonstrating homeostatic control mechanisms to affect tumor cell outgrowth in MTCL (Newrzela et al. 2008, Newrzela et al. 2012).