Abstract
Crohn's disease (CD) is a chronic, immune-mediated inflammatory bowel disease (IBD) with no known cure, resulting in significant morbidity. Goals of therapy include resolution of symptoms and mucosal healing. However, many patients have sub-optimal responses to currently available therapies. This represents a significant unmet medical need. Cell based therapies provide a powerful new tool to modulate the immune system without the harmful side effects associated with immunosuppressant medications. With the advent of cell-based therapies, there comes a need to better understand the molecular mechanisms of immune cell dysfunction in inflammatory bowel disease in order to develop powerful personalised therapies.“Thymus-derived” regulatory T cells (Treg) are effective in modulating immune responses in many pre-clinical models of IBD. There is recent data to show that Tregs purified from peripheral blood (PB) of CD patients play a critical role in controlling both phenotype and expansion of auto-reactive T cells. These autologous ex vivo expanded Tregs can be expanded to therapeutic numbers under GMP conditions while maintaining their suppressive ability and phenotypic stability due to treatment with Rapamycin. It is recognised that Tregs are needed at the site of inflammation. However, Rapamycin treated ex vivo expanded Tregs express minimal amounts of the gut homing molecule integrin α4β7. Retinoic Acid (RA) regulates the expression of the primary gut homing integrin, α4β7.
This thesis will demonstrate that retinoid treatment effectively induces the expression of α4β7 on ex vivo expanded Tregs (when compared to cells cultured under standard conditions (RAPA), cells cultured under standard culture conditions but with the addition of RAR568 (Rapa+RAR568) expressed significantly more α4β7 (95.9 ± 1.93 vs 5.947 ± 3.18, p<0.0001. These expaned Treg maintain their phenotypic stability and suppressive ability (cells expanded in the presence of RAR568 express high levels of FOXP3 (96.99% ± 3.51). This value is not significantly different to cells expanded under standard conditions (96.03 ± 6.18), they also suppress proliferation of 99.8% autologous Teff in a standard suppression assay). Furthermore, it highlights the need for selective targeting of the Retinoic Acid receptor α for highly effective induction of α4β7, whilst avoiding the all trans Retinoic Acid (ATRA) induced capacity to skew to a pro inflammatory cell phenotype. The functional relevance of α4β7 induction is highlighted by both in vivo and in vitro functional studies. During in vitro flow chamber experiments, a significantly higher proportion of RAR568 treated Treg were observed to engage with MadCAM and progress through stages of cell migration (p<0.0005). In an in vivo trafficking study which utilised a humanised mouse model, where animals were xenografted with human foetal small bowel, a significantly higher proportion of RAR568 treated Treg were seen in the inflamed xenografts compared with Treg expanded under standard conditions (p=0.0095).
To better understand the molecular mechanisms of Treg dysfunction, the pathogenic Crohn’s disease associated IL2Rα SNP rs61839660 was studied. Crohn’s disease patients bearing the TT, CT and CC genotype were recruited from the National NIHR IBD bio-resource, functional and phenotypic studies using mass cytometry (Cytometry Time of Flight; CyTOF) were performed on their peripheral blood mononuclear cells (PBMCs). Contrary to currently published reports, this SNP does not lead to loss of Treg function. In the TT genotype, it leads to increased expression of IL2Rα, most profoundly on effector T cells (Teff) ( MFI of CD25 on Tconv of CD patients homozygous for rs61839660 (TT) compared to those who are CT or CC (881.0 ± 89.30 vs 475.4 ± 59.19, p=0.0014; 881.0 ± 89.30 vs 463.3 ± 61.78, p=0.0012)) and subsequently phosphorylation of STAT5 at doses of IL-2 which only activate wild-type Tregs (TT Tconv had a higher proportion of pSTAT5 positive cells compared to CC (25.91 ± 6.514 vs 4.652 ± 1.547, p= 0.0052), leading to functional Teff hyper-responsiveness. This is confirmed by a more activated, gut homing phenotype seen on Teff of TT patients using CyTOF analysis (Treg and Tconv of TT patients express more GPR15 (p<0.005), CXCR3 (p<0.05) CD120b (p<0.005) than their CC counterparts0 .
Taken together, the work of this thesis has allowed for the development of an ex vivo expansion protocol of gut specific regulatory T cells for the purposes of a Phase 1 trial of Treg therapy in Crohn’s disease. The findings from the work with rs61839660 will allow for more comprehensive screening of patients enrolled into Treg cell-based therapy studies as well as trials of low dose IL-2 – which are currently under way for multiple autoimmune conditions. They have also brought us a step forward in understanding some of the molecular mechanisms underlying the dysfunctional relationship between Treg and Teff in Crohn’s disease.
| Date of Award | 1 May 2019 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Graham Lord (Supervisor) & Jeremy Sanderson (Supervisor) |