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In Chapters 3 and 4, we reported the occurrence of the Vβ6- _{T cells in 3 out of 7 FVB/N and 3 of 4} C57BL/6 mice produced during the diversification of the Marilyn TCR CDR3β. We deduce that the codon optimisation applied during the de novo synthesis of the Marilyn TCR (CDR3β diversifying) sequence may have contributed to these observations. The stretch of at least 18 identical nucleotides within the J-segments of the WT Marilyn TCRα and β chains is thought to facilitate a possible homologous recombination-deletion process which resulted in the formation of the chimeric TCR chain comprising of a Vα connected to a Cβ domain (Im et al., 2014; Zhang & Sapp, 1999). Importantly, the skip mechanism and resultant hybrid TCR chain did not show any significant effects on the development and maintenance of a functional ‘αβT cell’ repertoire. Through PCR analysis of the Vβ6- retrogenic T cells, we found that the Vα and Cβ domains remained intact, and the fusion was focused at an amino acid substitution for aspartic acid in resulting Jα-Jβ segment. According to the IMGT database, this particular amino acid can be found in the same position within the FR4 region of other

142 J-segments (Lefranc, 2005). Hence, we believe this substitution to have minimal, if any, difference in the folding of the essential β-sheets that make up the connected Vα and Cβ structures.

Based on the dominance of this TCR chain when it occurs in the retrogenic mice (i.e. almost all T cells were Vβ6-_{), the TCR may have enhanced MHC recognition or signalling properties. This suggests a} potential selection advantage of this TCR chain or that it is able to outcompete the other diversified Marilyn TCRs. The novel TCR configuration has major differences from the regular αβTCR configuration. First, the antigen-binding site is a Vα-Vα dimer without any Vβ CDR structure. Second, the inter-domain interfaces are modified, whereby the natural Vβ-Cβ interface is replaced with a distinct Vα-Cβ interface. Similarly, the natural Vα-Vβ antigen-binding surface is replaced by a Vα-Vα interface. As shown by the three-dimensional model of the fusion TCR chain, there may subtle differences between the conformation and position assumed by the Vα domain in place of the WT Vβ domain (Figure 5.4). As the novel Vα-Cβ TCR chain is produced following a non-V(D)J recombination event, we do not know the stage(s) of T cell development when this event occurs. Indeed it could take place prior to the ‘window’ of V(D)J recombination within the DN thymocyte compartment and so remove the recombination cassette. It is likely the Vα-Cβ chain will associate with the pre-TCR through the conventional Cβ-pTα interface (Pang et al., 2010). Retrogenic mice produced directly with the novel Vα-Cβ TCR chain developed peripheral T cell repertoires (Refer to Chapter 5.2.4). We can therefore presume a novel configuration of the pre-TCR comprised of the Vα-Cβ fusion/pTα chain is expressed and is able to substitute for the WT pre-TCR in progressing through the β-selection checkpoint.Following gene rearrangement of the endogenous Vα-Jα genes at the DP stage, the fusion TCR chain is able to pair efficiently with TCRα. The fusion chain retains an intact Cβ domain, which provides a protein scaffold for the Vα domain and CDR loops, and allows inter-chain disulphide interactions with the endogenous TCR Cα domain (Richman et al., 2009). Despite alterations to the antigen-binding surface, the hybrid TCR-endogenous TCRα ‘heterodimer’ can recognise self-pMHC and direct progression past thymic positive and negative selection. Further, higher affinity engagement with pMHC-II on mTECs and thymic DCs can also induce FoxP3 expression and stimulate differentiation into nTregs (Hanabuchi et al., 2010; Proietto et al., 2008; Aschenbrenner et al., 2007). As a result, we identified a typical T cell repertoire in the peripheral lymphoid organs, with a higher frequency of CD4+ _{than CD8}+ _{T cells, and a sizeable population of regulatory T cells. This data also} indicates that the novel TCR dimer engages productively with pMHC-I and pMHC-II, which further challenges the notion that germline TCR V-segments and CDR loops have intrinsic preference to bind to specific MHC classes (Sim et al., 1996; DerSimonian, Band & Brenner, 1991; Jameson, Kaye & Gascoigne, 1990).

143 Importantly, the novel TCR dimer is capable of associating efficiently with the CD3 signalling apparatus, as indicated by the similar levels of CD3 and TCRβ expression levels in the splenic T cells. Similar to the T cell signalling complex in Marilyn and MataHari diversified TCRs, it is reasonable to assume that the extracellular and TM structures within the Cβ domain are conserved to facilitate the crucial interactions with the CD3 subunits. In particular, the TCR Cβ F-G loop which mediates association with CD3γɛ and regulates αβT cell development may be advantaged to explain the dominance of the Vβ6- _{T cell population (Wucherpfennig et al., 2010; Touma et al., 2006; Call &} Wucherpfennig, 2005; Call et al., 2002).

With a new Vα-Vα antigen-binding interface, we may expect the novel TCR dimer to exhibit a different docking orientation and affinity compared to the WT Marilyn TCR. Generally, the TCRβ interacts with the α1 helix of the MHC and the C-terminus of the peptide, whereas the TCRα binds primarily to the α2 or β1 MHC helices and the peptide N-terminus (Rudolph, Stanfield & Wilson, 2006; Garboczi et al., 1996). Further structural studies would provide valuable insight into the new docking mode applied by the Vα-Vα interface, whether the hybrid TCR chain assumes the place of a conventional TCRβ chain. It is possible that a lack of CDRβ loops may necessitate the aid of compensatory mechanism for ligand engagement or that the novel TCR CDR must undergo numerous and possibly large conformational changes upon pMHC binding (Burrows et al., 2010; Armstrong, Piepenbrink & Baker, 2008). However, the challenge of producing TCR-pMHC co-crystals here is that the specific antigen of this hybrid TCR is not yet clear. Since it only retains the Marilyn Vα1.1 segment, and binds to a wide array of endogenous TCRα chains, logic dictates that the TCR would not be restricted to the Marilyn-specific Dby complexed with H2-Ab_{. Conversely, minimal conservation of key germline contacts has been shown to retain} pMHC specificity in TCRs where the rest of the sequence remain unchanged (Scott-Browne et al., 2011). Functional studies must then be performed to investigate and identify the immunogenic ligands, including antigenic challenge with model antigens such as hen egg lysozyme (HEL) or ovalbumin (OVA; Kumar, Cristan & Paul, 2008). Preliminary proliferation assays (not shown) where retrogenic T cells expressing the chimeric TCR chain are cultured with syngeneic (C57BL/6) and allogeneic (FVB/N) spleen cells have demonstrated that the TCR retains pMHC specificity and can respond to foreign pMHC.

Overall, the study presented in Chapter 5 demonstrated the dispensability of the germline-encoded TCR CDRβ as well as the entire Vβ domain in mediating MHC-directed events such as T cell selection, signalling, development of nTregs and homeostatic proliferation. Such observations are not in line with the model which proposes germline CDRs have co-evolved with MHC over time and play key roles in contacting MHC chains and impose MHC restriction on αβTCR recognition (Garcia et al., 2012; Marrack et al., 2008). The tremendous plasticity of the TCR despite substitution of a Vβ with a Vα

144 domain without significantly affecting the ability to recognise MHC supports the idea that extrinsic factors enforce MHC restriction on the αβTCR during thymic selection (Tikhonova et al., 2012; Van Laethem, Tikhonova & Singer, 2012; Van Laethem et al., 2007). The TCR appears to employ an opportunistic antigen-binding interface, rather than being hardwired to engage MHC ligands (Holland et al., 2012; Attaf et al., unpublished).