Understanding the balance of immune-stimulatory and immune-suppressive antigen specific responses to SARS-CoV-2 antigens is crucial for gaining insight into COVID-19 pathology, as well as for designing effective vaccine strategies. T cell responses to the SARS-CoV-2 Spike protein have proven to be long-lasting and protective in some human vaccine clinical trials. The Enzyme Linked Immuno Spot (ELISpot) assay is a powerful and sensitive tool to study antigen specific functional T cell responses. By using the Fluorospot method of the ELISpot, which incorporates multiple fluorescent readouts, we can not only assess individual T cell reactivity but also distinguish between immune-stimulating and immune-modulatory or suppressive responses by simultaneously measuring IFN-γ and IL-10. Preference for IFN-γ or IL-10 induction to Spike antigens could affect the overall strength, and protective potential, of vaccine induced responses. T cell responses to Spike ancestral and variant peptide pools (including novel and cross-reactive T cell epitope regions) were optimised in healthy blood donors using duo-colour Fluorospot assay. Donor specific IFN-γ responses were observed to both CD8 and CD4 peptide pools. Surprisingly, there was a preferential IL-10 induction to specific CD4 T cell epitopes within the pools, suggesting an intrinsically immunosuppressive bias. Immunogenicity to Spike peptide pools were optimised in healthy blood bank donors, and further studies will examine vaccine induced T cell responses in the Platform Trial In COVID-19 vaccine BOOsting clinical trial (PICOBOO), which aims to evaluate the effectiveness, safety, reactogenicity and immunogenicity of different booster vaccination strategies against SARS-CoV-2 ancestral and variant strains in the Australian population. Understanding the vaccine induced immune response to optimised SARS-CoV-2 Spike peptide epitope pools, with an intrinsic biological IFN-γ or IL10 induction bias, will further our knowledge of antigen-specific and cross-reactive immune-modulation by SARS-CoV-2 antigens and help inform future booster vaccination strategies.