School of Biomedical Sciences - Theses

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    Investigating the requirements for robust CD4+ T cell immunity to Herpes Simplex Virus.
    Harpur, Christopher Mathew ( 2016)
    CD4+ T cell responses are crucial for the control and clearance of many intracellular pathogens, including Herpes Simplex Virus type 1 (HSV-1), yet the requirements for the induction of optimal effector responses are unclear. To better understand the stimuli required for the generation of sustained T cell immunity, we compared transgenic HSV-1-specific CD4+ T cell responses following epicutaneous, intranasal and intravenous inoculation of mice with UV-inactivated (UV-HSV) and live HSV-1. This thesis describes these observations and demonstrates that despite comparable early proliferative responses, HSV-1-specific CD4+ T cells increased T-bet expression and accumulated in mice infected with live HSV, but not following inoculation with UV-HSV. Phenotypically, HSV-1-specific CD4+ T cells activated by UV-HSV in vivo lacked typical effector cell characteristics including CD25, a component of the high affinity IL-2 receptor, which promotes T cell expansion, survival and Th1 differentiation. This was found to occur despite the prolonged persistence of UV-HSV-derived antigen during early priming, a known requirement for CD4+ T cell differentiation. Dividing CD25-, CD4+ T cells elicited by UV-HSV resembled those that were adoptively transferred into mice at a late stage of an acute epicutaneous infection when virus in the skin had cleared and antigen levels had presumably waned. Congruent with this, infection with an attenuated mutant HSV-1, incapable of more than one replication cycle, elicited a proportion of CD25+ CD4+ T cells significantly greater than and less than that generated by UV-HSV and wildtype HSV-1, respectively. These findings suggest that HSV-1 antigen abundance during T cell priming is vital to generate a sustained CD4+ T cell response. To resolve whether these disparate priming patterns were due to differences in how dendritic cells (DC) stimulate CD4+ T cells in response to live and UV-HSV, DC were isolated from the lymph nodes draining the site of infection and assessed their ability to stimulate HSV-1-specific CD4+ T cells ex vivo. Intriguingly, although both CD8α DC and migratory dDC presented virus-derived antigen on MHC II molecules following live virus infection, presentation of UV-HSV-derived antigen was largely confined to the dDC with very little, if any contribution by the CD8α DC. The reduced contribution by CD8α DC in UV-HSV-inoculated mice also correlated with a lack of CD40 up-expression, a DC maturation marker, from this subset. Consistent with the view that epicutaneous UV-HSV inoculation failed to convert naïve HSV-1-specific CD4+ T cells into T-bet expressing effectors cells because CD8α DC did not participate in the priming, live virus infection of mice lacking CD8α DC resulted in similarly impaired HSV-1-specific CD4+ T cells priming characterized by reduced T-bet expression. Finally, supplementation of epicutaneous UV-HSV administration with targeted HSV-1 antigen to CD8α DC was found to profoundly augment T-bet expression in respondent CD4+ T cells. Collectively, these data implicate lymph node-resident CD8α DC as a source of critical Th1 differentiation cues and propose a functional rationale for recently reported DC:CD4+ T cell dynamics during CD4+ T cell induction in response to skin infection with this pathogen.