THE OT-II MOUSE AND POSSIBLE FUTURE EXPERIMENTS.

Towards the end of this study, the OT-II mouse was procured with a view to

investigating interactions between antigen presenting eosinophils and antigen-specific T cells. Initial experiments suggested that eosinophils could present antigen to OT-II T cells, however no detectable Th2 cytokine production could be measured despite specificity of the transgenic TCR for the Th2 skewing OVA323-339 peptide (Janssen et

a mouse model of allergic airways inflammation, and was found to be refractory to disease induction, which was attributed to significant IFN-γ release from T cells in response to stimulation with OVA (Chapter 6).

T cell therapies have been effectively used to restore anti viral responses in immunocompromised patients (Heslop et al., 1996; Rooney et al., 1998), and the infusion of opposing Th1 cells in order to attenuate Th2 mediated allergic disorders is an exciting prospect. For instance, the adoptive transfer of antigen-specific Th1 cells

that secrete IFN-γ has previously been shown to attenuate characteristics of allergic airways disease in animal models, including reduced pulmonary eosinophilia and airways hyperresponsiveness (Cohn et al., 2001; Huang et al., 2001). Subsequent

attempts at using adoptively transferred IFN-γ secreting OT-II CD4+ T cells to reduce allergic airways disease in C57BL/6 mice gave mixed results. While the recruitment of eosinophils to the airways lumen was certainly inhibited, recruitment to lung tissue, airways hyperresponsiveness and OVA specific immunoglobulin were left intact. This suggests that such immunotherapies based on homogenous populations of T cells with opposing cytokine responses to allergen may be of little therapeutic value, although it is acknowledged that using cells derived from transgenic animals that only recognise a single T cell epitope may not be an ideal foundation on which to predict future results.

The availability of antibodies directed against the OT-II TCR Vβ5 element enabled a brief and interesting investigation into the effects of neonatal antigen administration, which has previously been shown to inhibit both Th1 and Th2 responses (Hogan et al., 1998a). Soluble OVA administration to OT-II neonates was shown to modulate adult responses to sensitisation and challenge with OVA, and led to the restoration of pulmonary eosinophilia, Th2 cytokine production, and OVA-specific IgG1 to levels

equivalent to those seen in OVA sensitised and challenged wild type mice. In Chapter 7, it was suggested that antigen delivery to OT-II neonates reprogrammed circulating T cells in the periphery during ontogeny, and that these cells were responsible for later development of allergic airways disease in OVA sensitised and challenged adults. It is reasonable to think that subsequent de novo production of TCR transgenic T cells in OVAN treated OT-II adults may still lead to mature T cells exiting from the thymus

with the observed phenotype (IFN-γ secretion) seen in unmanipulated OT-II mice. Therefore, it is quite possible that in OVAN treated OT-II mice, subsequent i.p.

sensitisation and challenge with OVA led to the suppression and/or immunomodulation of these recent de novo produced thymic emigrants, by the Th2 type TCR transgenic cells (and their descendents) that were reprogrammed in the neonatal periphery. Future experiments using elispot assays and individual peripheral CD4+/Vβ5+ DP T cells may confirm the presence of two distinct populations of TCR transgenic T cells in the

unsensitised OT-II OVAN mouse: one population that may secrete IFN-γ in response to OVA and which is representative of recent thymic emigrants, and an older population comprising those peripheral T cells and their descendents that were reprogrammed in the neonatal periphery, and which secrete IL-5 and IL-13 in response to OVA.

This question remains: why did a large soluble dose of OVA delivered to OT-II neonates not induce the previously observed anergy seen in wild type mice (Hogan et al., 1998a)? In the wild type mouse, maturing thymocytes specific for OVA peptides may only represent a small fraction of all developing thymocytes. Administration of soluble OVA may have led to the negative selection of those cells that were developing in the thymus at that time (Liblau et al., 1996), and the retention of OVA peptides by thymic medullary APCs may have prolonged the negative selection of the subsequently developing OVA-specific T cells. The (few) mature OVA-specific T cells in the wild type neonatal periphery may have been overwhelmed by such a large dose of soluble antigen and been subject to clonal exhaustion and apoptosis (Boehme and Lenardo, 1993; Radvanyi et al., 1993; Nagata and Golstein, 1995; Iezzi et al., 1998). In the OT-II neonate, a disproportionately large number of OVA-specific TCR transgenic T cells may be expected to reside in the periphery, and these cells may have been impervious to such an overwhelming dose of soluble antigen by virtue of their sheer numbers, and possibly synchronised responses afforded by the possession of a common TCR. In Figure 8.2, a mechanism is proposed wherein OT-II CD4+ T cells may be

reprogrammed during ontogeny. Future experiments may reveal that OVA

administration to OT-II neonates can promote the induction of allergic inflammation of the lung (when subsequently exposed to inhaled allergen) without the requirement for sensitisation to OVA.

In summary, the availability of the OT-II mouse has provided a brief but unique

Chapter 7 Neonatal Antigen Delivery Induces Th-2 Mediated Allergic Airways Disease In The OT-II Transgenic Mouse High dose soluble

OVA administration to neonates OVA/Alum OVA Aerosol Diverse CD4+ T cell repertoire - consists of already mature cells in periphery. Low frequency of OVA-specific cells. Anergic/depleted OVA-specific T cell population. Refractory to AAD. OVA/Alum OVA Aerosol OVA-specific CD4+ T cell population - consists of already mature cells in the noenatal periphery Th2 TCR transgenic cell population. AAD induction D AAD progressionD -ve selection of

OVA specific cells in thymic medulla -ve selection of TCR transgenics in thymic medulla Clonal Exhaustion? No emergence of OVA-specific T cells from thymus for some time. Other TCRs not affected

No emergence of TCR transgenic T cells from thymus for some time

Re-programming of OT-II TCR transgenics towards Th2 - abilty to secrete IL-13 and IL-5 in response to OVA.

Figure 8.2. Proposed mechanism for reprogramming in the OT-II neonate exposed to a high dose of soluble OVA. In the wild type neonate, a high dose of soluble OVA (WILD TYPE OVAN) may promote the negative selection of OVA-specific thymocytes, and this process may continue into adulthood due to the retention of OVA peptides by medullary APCs. In the wild type periphery, the few recent thymic emigrants that are OVA-specific are overwhelmed by the large amount of soluble antigen, and either die or become anergic through clonal exhaustion. The wild type OVAN mouse is therefore refractory to allergic airways disease (AAD) induction and progression, and Th1 responses are also attenuated. In the OT-II neonate, negative selection in the thymus may also occur, and may also continue well into adulthood. There are many OVA-specific TCR transgenic T cells in the periphery, which are not overwhelmed by large amounts of soluble antigen. Instead, they may be collectively reprogrammed towards the Th2 phenotype. This promotes AAD induction and progression in OVAN treated OT-II mice.

Future experiments may illuminate the mechanism for reprogramming of the transgenic T cell during the neonatal period, and further our understanding of neonatal immunity.

Chapter 9