Immune Cell Activation

In the area of immune cell activation, our lab has focused on T cells. These cells originate in the bone marrow and mature in the thymus, where only a small number survive the process of maturation. Thymic education is responsible for self-recognition and central tolerance. T cells share structural features of the T cell antigen receptor. A peculiarity of the T cell receptor is that it requires foreign antigens to be presented in the context of self; that is, the cells only see antigen when it is complexed with a molecule from the major histocompatibility complex (MHC). For many years, we have wondered what keeps normal T lymphocytes from being activated all the time. After all, we are constantly bombarded with antigens. Initially, we focused on signals that activate the cell cycle machinery (see Figure 1). The conventional wisdom then, and until a few years ago was that, because T cells are highly specific (they only recognize one antigen among the millions possible), they would remain quiescent until they saw their precise antigen and would be driven through the cell cycle by pathways that regulate the cell cycle machinery. Specifically, activation of CDK4 was a central mediator of the transition from the G0 to the G1 phase (see Figure 2). But the dilemma was that no other cell in the body seemed to behave this way. In fact, our work showed that the machinery that controls proliferation in lymphocytes was similar to that present in other cells (remarkably, this is similar to the machinery that controls cell division in all eukaryotic organisms, including yeast!) In the latter 1990’s and the early 2000’s, several groups proposed that, in fact, T lymphocytes were not unlike other cells, and that they were quite ready to be activated at any time. However, this activation was kept in check by specific proteins that were called “negative regulators” (see Figure 3). We pursued this concept of negative regulation, and we showed, in collaboration with Dr. Baksh and Dr. Burakoff at Harvard Medical School, that a protein called NFATc2, which people previously recognized as an “activator” also was important to maintain this negative regulation in T lymphocytes (see Figure 4). This work was published in a prestigious journal called “Molecular Cell” in November of 2002. We followed this lead, and our recent work shows that one of the ways that smoking can affect the immune system, and probably contribute to many of the diseases associated with tobacco use, is by nicotine “enforcing” negative regulation - that is, it prevents T lymphocytes from responding to foreign invaders - through the action of this NFATc2 protein. This work, published in the Journal of Pharmacology and Experimental Therapeutics in October of 2004, was mostly led by Dr. Ashley Frazer-Abel, a senior post-doc in the lab who recently accepted a faculty position as Instructor of Medicine at the University of Colorado Health Sciences Center. Our lab continues to explore the mechanisms that control T lymphocyte activation, as well as how nicotine or tobacco products affect the immune system with a renewed focus on the role of cholinergic receptors, MHC antigens, and other molecules in negative regulation.

Figure 1. Direct correlation between CDK4 expression and activity and cytokine-responsiveness in primary T cells. CDK4 gene expression (A, E), CDK4 protein accumulation (B, F), CDK4 kinase activity (C, G) and competence to proliferate in response to IL-2 stimulation (D, H) were measured in resting peripheral blood T cells (U/S), T cells stimulated with a mitogenic concentration of PHA (10 µg/ml, PHA-MITO), T cells stimulated with submitogenic PHA (0.5 µg/ml for 1 hr, PHA-COMP), or T cells stimulated with submitogenic PHA in the presence of herbimycin A (3 µM, H.A.) or staurosporine (10 nM, ST) as indicated. Expression of the CDK4 and ß-actin genes was assessed by RT-PCR 5 hrs after stimulation; cell lysates were prepared 15 hrs after stimulation for immunoblotting and for immunoprecipitation of CDK4 complexes for in vitro kinase activity assays by phosphorylation of a truncated recombinant Rb protein (p56/Rb); and IL-2-responsiveness was determined by 3H-thymidine incorporation 48 hrs after stimulation. Twelve donors showed the “resting” phenotype shown in panels A-D. The donor shown is representative of data from 3 (A), 9 (B), 3 (C), and 11 (D) individuals, respectively. Five donors showed the “pre-activated” phenotype shown in panels E-H. The donor shown is representative of data from 2 (E), 3 (F), 2 (G), and 5 (H) individuals, respectively. Densitometric data provided under the immunoblots and kinase autoradiographs are normalized to a level of 1.0 present in competent T cells. Equivalent loading was confirmed by staining gels after transfer and membranes after immunoblotting with Coomassie blue. From Modiano et al, J. Immunol. 165:6693, 2000.

Figure 2. Cdk4 is a master regulator of cell cycle progression in human T cells. In order to engage resting T cells to enter the cell cycle, a signal from the environment (antigen) must activate Cdk4. Partial activation by the antigenic signal allows the cell to progress through the G0/G1 transition into a “competent” state, where the cell can respond to a second signal delivered by a growth-promoting cytokine like IL-2. In the presence of the cytokine signal, the cell will progress through G1 and into S phase, where DNA is replicated and the cell can eventually divide. If there is an incomplete signal, or if the cell receives a growth-inhibitory signal, then it enters a state of “anergy” or unresponsiveness. Various signals can also lead the cell to undergo apoptosis, which is regulated by processes that are most likely independently from Cdk4 activity.

Figure 3. Events that control cell cycle entry and G1 phase progression in T cells. Resting T cells are actively maintained the G0 phase of the cell cycle by negative regulatory molecules (shown in red). Inactivation of these negative regulators to allow cell cycle progression requires appropriate stimulation to promote transition from the G0 to the G1 phase and to prevent apoptosis. Non-linear biochemical cascades lead to specific events that promote survival and transition to the G1 phase. Expression and activation of CDK4 are critical events at this stage of T cell activation. Progression through the Restriction point “R” of the G1 phase and transition to the S phase are similarly dependent on the orderly occurrence of biochemical events, including sustained activity of CDK4 and CDK2 that inactivate Rb and promote transition to the S phase.

Figure 4. Cdk4 is subject to negative regulation. This updated model shows how NFATc2, a transcription factor that negatively regulates T cell activation works to inhibit Cdk4 expression. NFATc2 is only present in small amounts in naïve resting T cells, so it probably contributes minimally to maintain quiescence in these cells. However, activated T cells express high amounts of NFATc2, and it seems to be an important factor that drives antigen-experienced or memory T cells back into the G0 phase (or a resting state). NFATc2 also seems to be necessary to induce cytokine-unresponsiveness in the presence of growth inhibitory signals, such as those delivered by nicotine.