Potent Signal 1 as a noncanonical Signal 3: antigen location, and peptide:MHCII complex density and half-life as drivers of CD4+ T cell differentiation - PROJECT SUMMARY CD4+ T cells (TCD4) play critical roles in adaptive immune responses following antigen-driven proliferation and differentiation. By convention, proliferation is driven by two triggers: “Signal 1” in the form of peptides (epitopes) derived from extracellular antigens and displayed at the surfaces of antigen-presenting cells (APCs) by MHC class II molecules (MHCII), and “Signal 2” in the form of co-stimulatory molecules expressed by APCs in re- sponse to pathogen associated motifs (“stranger/danger”). Convention also holds that TCD4 differentiation is driven by cytokine environments at sites of activation (“Signal 3”), resulting in the emergence of one or more functional TCD4 subsets (TH1, TH2, TH17, Treg and/or TFH). Canonical Signal 3 is clearly important, but it is well established that strength of Signal 1 (“antigen dose”) can also govern TCD4 differentiation. However, studies making this point have largely involved purified proteins or synthetic peptides in in vitro settings, with outcomes varying widely with respect to the functionalities associated with low and high antigen doses. Extension of our work on MHCII-restricted presentation of influenza (flu)-derived epitopes could provide clarity in this regard. Specifically, we have established that MHCII epitopes can be generated not only from extracellular (“exogenous”) antigen but also intracellular (“endogenous”) antigen following antigen expression within the APC, and that en- dogenous vs. exogenous antigens drive far more potent TCD4 responses, with respect to expansion and, per preliminary data, functionality. Based upon these and other findings reviewed below, we propose the following model: 1) Extracellular antigens in various forms, not necessarily presenting a threat, are inefficiently converted to peptide:MHCII (p:MHCII) complexes via conventional (endosomal) antigen processing (a weak Signal 1), driv- ing limited TCD4 expansion and functionality. We define functionality as the average number of effector functions (cytokine production and cytolytic capacity) independent of subsetting conventions. 2) Abundantly produced en- dogenous antigens, reflecting active infection, lead to dense and sustained p:MHCII display at the APC surface (strong Signal 1). This strong Signal 1, signaling a potential threat, drives robust TCD4 expansion and functionality, thereby enhancing the TCD4 response and the prospects for a successful defense. Grounding our studies in mRNA-based vaccination, in combination with several technical innovations, we will test this model by: a) varying antigen location via microRNA-based targeting of antigen-encoding mRNA (Aim 1), and b) modulating strength of Signal 1 with respect to both density and half-life of surface peptide:MHCII complexes while holding other key parameters constant (Aim2). Impact on TCD4 functionalities will be assessed via flow cytometry and protection studies. The proposed studies could fundamentally revise the working models of threat perception and TCD4 differentiation, motivating novel approaches to modulating TCD4 in settings where their participation is advanta- geous or deleterious.
