Studies On The Roles Of Intracellular Ca2+ And Reactive Oxygen Species During CD4+ T Cell Activation : Influence Of Signal Strength

Abstract

Optimal CD4+ T cell activation is key to the generation of a productive immune response. Naïve circulating CD4+ T cells are quiescent under normal conditions and undergo activation only upon encounter of the T cell receptor (TCR) with Major Histocompatibility Complex (MHC)-encoded class II molecules on antigen presenting cells (APCs). Processed antigens (derived from pathogens, tumors or self tissue during autoimmunity) in complex with MHC class II are recognized by specific TCRs on CD4+ T cells. After this encounter, the highly complex and regulated process of CD4+ T cell activation results in the differentiation of naïve T cells into effectors and their clonal expansion. Apart from binding to its cognate peptide-MHC-II complex, several other factors define the extent and magnitude of T cell activation. This context is an important determinant of the nature of the subsequent T cell response. One of the factors involved is the strength of the signal (SOS) which is delivered to the cell upon ligation of the TCR to the MHC-peptide complex. The SOS, which can vary from weak to strong, is determined by the affinity/avidity of the TCR for the MHC-peptide complex, antigen concentrations, the duration of engagement, etc. Extreme weak or strong signals can lead to non-productive T cell responses with the former resulting mostly in anergy and the latter in cell death. Signals of optimal strength are the ones that translate into functional T cell responses. However, mechanisms by which signal strength information is translated into distinct T cell responses are still not very well understood. Binding of the TCR to the MHC-peptide complex triggers several signaling cascades and leads to generation of intracellular signaling intermediates, including Ca2+. Rise in intracellular Ca2+ levels is one of the first events to occur upon initiation of T cell activation. The initial increase is brought about due to release of Ca2+ from intracellular smooth endoplasmic reticulum stores. Once intracellular stores have been emptied, the increase is sustained by a process termed as capacitative Ca2+ entry, involving opening of Ca2+ channels in the plasma membrane known as Ca2+ release activated channels (CRACs). Consequently, Ca2+ flows from the extracellular milieu into the cell. A sustained Ca2+ increase is essential for activation of the transcription factor, NF-AT whose primary job is to initiate transcription of IL-2, a cytokine crucial for CD4+ T cell proliferation. The other intracellular signaling intermediates which are the focus of work presented in this study are reactive oxygen species (ROS). TCR signaling leads to generation of ROS, which may be either mitogenic or detrimental to T cell activation. Low levels of ROS, especially H2O2, inactivate phosphatases leading to activation of kinases and signaling pathways resulting in increased proliferation. However, high levels of ROS cause oxidative stress leading to reduced T cell activation, hyporesponsiveness and death. The experimental system used for this study consists of purified mouse lymph node CD4+ T cells. These cells were activated with varying strengths of the primary signal to better understand the roles of Ca2+ and ROS in modulating T cell activation and function. The signal strength was either varied by addition of different concentrations of ionomycin or thapsigargin, pharmacological agents that increase intracellular Ca2+ concentrations. Alternatively, signal through the surface TCR-CD3 complex was initiated using anti-CD3 in two modes: soluble (weak signal) or plate immobilized (strong signal). Increasing concentrations of ionomycin or thapsigargin or changing the mode of anti-CD3 from soluble to plate bound enhances IL-2 amounts, thereby converting a weak signal to a strong one. The work presented has been divided into three parts, each dealing with a distinct aspect of T cell activation. I. SOS and CTLA4-CD80/CD86 interactions: The binding of the TCR to its cognate MHC-peptide complex delivers the primary signal. However, this alone is not sufficient to drive T cell activation and an additional costimulatory signal emanating from the binding of CD28, a constitutively expressed receptor on T cells, to its ligands CD80 and CD86 is required. Another receptor that binds to CD80 and CD86 is CTLA-4 although it does so with a ~100 fold higher affinity. CTLA-4, unlike CD28, is expressed upon T cell activation and is considered to downregulate T cell activation. Its role as a negative regulator is highlighted by the phenotype of Ctla4 -/-mice which die of massive lymphoproliferation. However, there have also been reports of some plasticity in the effects mediated by CTLA-4. Previous work from our laboratory showed that CTLA-4-CD80/CD86 interactions could either inhibit or stimulate T cell activation depending on the SOS. To identify the molecular mediators of the differential effects of CTLA-4, the role of Ca2+ and ROS was evaluated. During activation with phorbol myristate acetate (PMA) and low amounts of ionomycin, intracellular amounts of Ca2+ were greatly reduced upon blockade of CTLA-4-CD80/CD86 interactions. Further experiments demonstrated that CTLA4-CD80/CD86 interactions reduced cell cycling upon activation with PMA and high amounts of ionomycin or thapsigargin (strong SOS) but the opposite occurred with PMA and low amounts of ionomycin or thapsigargin (weak SOS). These results were confirmed by activating cells with anti-CD3 either in the soluble or plate bound form. Considerably higher amounts of intracellular Ca2+ were present in cells activated with plate bound anti-CD3 compared to those activated with soluble anti-CD3. These amounts, further augmented by CTLA-4-CD80/CD86 interactions, probably became toxic to cells as increased proliferation was observed, using reagents that blocked these interactions. The opposite, however, was seen in cells activated with soluble anti-CD3. Also, CTLA4-CD80/CD86 interactions enhanced the generation of ROS. Studies with catalase revealed that H2O2 is required for IL-2 production and cell cycle progression during activation with a weak SOS. However, the high amounts of ROS produced during activation with a strong SOS reduced cell cycle progression. Together, this study identifies intracellular Ca2+ and ROS to play important roles in the modulation of T cell responses by CTLA4-CD80/CD86 interactions. II. SOS and CD4 downregulation: This study was initiated to identify early T cell functional responses that would help predict the strength of the primary signal. Using the in vitro culture system of varying signal strengths, it was found that CD4 surface expression was controlled by signal strength. CD4 is a surface glycoprotein expressed on the TH subset along with the TCR. It performs two main functions: First, binding to MHC class II and strengthening the TCR-MHC interaction, i.e. functioning as a coreceptor. Second, due to its association with p56lck a src family tyrosine kinase, the presence of CD4 along with the TCR enhances signal transduction. Also, CD4 acts as a receptor for entry for the AIDS virus. It is known that CD4 is downregulated from the surface and degraded upon T cell activation by a protein kinase-C dependent process in human and mouse T cells. Experiments presented in this study showed increased CD4 downregulation with a strong signal. The roles of intracellular mediators were assessed and high intracellular Ca2+ amounts, but not PMA activation, was required for sustained CD4 downregulation. Also, increased H2O2 amounts in cells activated with a strong SOS inhibited CD4 downregulation. Most interestingly, the pattern of CD4 downregulation was different between peripheral T cells and thymocytes, suggesting a correlation with CD4+ T cell development. III. Modulation of CD4+ T cell activation by small molecule plant growth regulators: An important area of investigation in T cell biology is the identification of molecules that modulate T cell activation. Towards this end, the mechanisms by which small molecule plant growth regulators, naphthalene acetic acid (NAA), 2,4 dichlorophenoxyacetic acid (2,4D) and indole acetic acid (IAA), influence CD4+ T cell activation was studied. It is useful to recall that IAA is the natural auxin present in plants, NAA is a synthetic auxin and 2,4D is a herbicide. These compounds, but not structurally similar control molecules, increased the activation and IL-2 production in CD4+ T cells activated with either soluble anti-CD3 or a combination of PMA and ionomycin. An investigation into the mechanisms of action by these compounds revealed increased early generation of intracellular ROS and Ca2+. Interestingly, the nature of their effects was found to rely on the strength of the primary signal: IL-2 and proliferation were increased in cells activated with a weak signal, but lowered proliferation was observed in cells activated with a strong signal. Cells activated with strong signal posses high amounts of ROS and Ca2+ and further increase in their amounts by IAA, NAA and 2,4D resulted in growth suppression. However, augmentation of Ca2+ and ROS amounts in cells activated with a weak signal was mitogenic. The role of these compounds during in vivo T cell responses needs to be addressed. Taken together, results presented in this study emphasize the importance of the role of SOS in determining T cell responses. In addition, the roles of Ca2+ and ROS downstream of the primary signal in modulating CD4+ T cell activation were demonstrated.