oa CD4+T Cells Are Programmed to Differentiate Before Entry into Division

In order to provide a protective host response to a vast array of invading pathological microorganisms the ability of naïve CD4⁺T cells to differentiate into discrete effector subsets, each able to mediate specific aspects of immunity is a central tenet of the adaptive immune response. T helper (Th) 1 cells mediate protection against intracellular pathogens, such as bacterial and viral infections, whereas Th2 cells mediate protective responses against extracellular pathogens such as helminthic parasites. Additionally, naive T cells may be induced to differentiate into T regulatory (Treg) cells, with Treg cells serving as an immunological checkpoint to dampen the immune response and protect against inappropriate activation of the immune system. As in the case of autoimmune diseases where aberrant Th1 cell differentiation occurs in response to self antigens or in the case of asthma and allergic responses where aberrant Th2 cell differentiation occurs in response to non-self environmental antigens. In order to activate naïve T cells and induce them to differentiate to combat a specific invading pathogen dendritic cells (DC), which are present throughout the body serving as cellular sentinels constantly surveying there surrounding for evidence of infection must be activated. Activation of DC occurs through ligand mediated activation of pathogen associated molecular pattern receptors or danger associated molecular pattern receptors. Allowing for the engagement of specific downstream patterns of effector molecule regulation that play an instructive role in the decision making process which occurs during the activation, division and differentiation of naïve T cells. Current dogma suggests that the generation of differentiated effector CD4⁺T cells takes place over a 3–4 day period following the initial engagement of the T cell receptor (TCR). Activation of CD4⁺T cells is thought to occur in three distinct functional phases, priming, proliferation and differentiation. Through the use of an in vitro culture system that allows for precise control of the factors which regulate the activation of naïve CD4⁺T cells we initially assessed the requirements for cytokine signaling vs. strength of TCR signaling during differentiation. Here, we determined that Th2 differentiation can be driven following activation with a weak TCR stimuli in the absence of additional cytokine inputs, indicating that Th2 differentiation occurs through a default endogenous pathway following activation. Whereas, Th1 differentiation required both a strong TCR signal and the presence of an instructive cytokine, in this case IFNg in order for differentiation to occur. We additionally investigated the kinetics of upregulation of the master transcription factors that regulate commitment to a specific T-helper phenotype. CD4⁺T cells acquire the disposition to commit to either Th1, Th2 or Treg lineages very soon after activation >24 hr and prior to the induction of division, as evidenced by increased expression of the master transcription factor proteins Tbet, GATA3 or Foxp3. Further, we show that entrance into cellular division is not required for the induction of a full program of differentiation to occur. By interrupting the activation of naïve CD4⁺T cells at different time points following stimulation through the use of anti-MHCII antibody. We were able to probe the effects that both alteration of TCR signal strength and alteration of TCR signal length have on the induction of differentiation as compared to division. This approach allowed us to demonstrate that TCR signaling is responsible for two distinct activation programs, whereby the strength of signal that a naïve T cell receives working in concert with or without cytokine at an early phase can induce Th1 or Th2 differentiation respectively. Whereas the length of the TCR signal can be construed as a secondary component of activation which controls the ability of CD4⁺T cells to enter into division. Here, we determined that at low concentrations of antigen TCR signaling must occur for 2 hr. Demonstrating that the signal delivered through the TCR represents not only an essential component for inducing an immune response through the initial activation of naïve CD4⁺T cells, but also acts as a rheostat that is able to determine both the strength and the length of TCR signal received. In turn controlling cellular decision making by dictating the outcome of differentiation and whether a cell will enter into cycle. As such these results have important implications for the rational design of vaccination strategies, in order to modulate components of TCR signaling cascade to direct an optimal response against the target vaccine antigens.