Functional Biology Of T Cells

National Institute Of Allergy And Infectious Diseases

Investigators

Linked publications & trials

Abstract

T lymphocytes play critical roles in immune defense against viruses, bacteria, fungi, protozoa, and cancer cells. Upon encounter with antigens on dendritic cells, these resting T-cells differentiate into effector cells that leave the lymphoid tissues and blood, entering sites of infection to combat pathogens. They can also cause autoimmune pathology. Most activated T-cells die, but some remain as memory cells, with some in lymphoid compartments and others patrolling peripheral tissues. Other lymphocytes such as regulatory T cells contribute to suppression of these T-cell responses. T cells also play a central role in the regulation of B cell antibody responses. This project attempts to gain both a qualitative and quantitative understanding of the activation, differentiation, migration, cell-cell interaction, memory status, and reactivation properties of both CD4 and CD8 T-cells and their interactions with other immune cells types such as dendritic cells and B cells. Through this research, a better understanding of lymphocyte dynamics and tissue architecture during an immune response to infection or after vaccination or during an autoimmune response will be established. These new insights can contribute to the more effective design of vaccines, to strategies for the amelioration of autoimmune processes, and for immunotherapy of cancer. These various in situ studies will be complemented by in vitro experiments examining details of the molecular control of T cell activation and the effects of costimulatory and negative regulatory pathways. We previously used Histo-cytometry to discover that pSTAT5+ Treg cluster around mostly migratory dendritic cells together with Tconv cells at comparable numbers in germ-free and conventional SPF mice, indicating that the activation of Tconv to the IL-2 producing state involves self-recognition. We have now used our advanced imaging platforms to determine the fate of the auto-activated CD4 T cells whose potential for tissue damage is controlled by the co-clustering Treg. In addition to IL-2, activated self-responsive Tconv cells express PD-1. Using this marker, a highly quantitative spatial statistical analysis revealed that Tregs are concentrated in micro-domains around the PD-1+ Tconv. This accumulation depends on IL-2 from the Tconv cells and involves local proliferation of the Tregs, which also undergo conjoint TCR and IL-2 driven activation to become effector Tregs. These activated Tregs have high CD25 and CTLA-4 expression, which minimizes IL-2 production and CD25 expression by the Tconv cells, resulting in efficient IL-2 stealing by the CD25hi Tregs. The TCR activated Tconv cells nevertheless enter cell cycle, but without access to IL-2 they rapidly die, pruning the repertoire of these autoreactive cells. A mathematical model shows an intricate, non-linear combination of factors involving Treg limitation of co-stimulatory signals, consumption of IL-2, disruption of CD25 upregulation, and cytokine deprivation enable Tregs support of immune homeostasis, with even 50% changes in CTLA-4 expression or Treg density allowing escape of Tconv cells from control. These studies have been made possible through use of Ce3D tissue clearing, which permits examination of physiologically small numbers of cells in entire lymph nodes. Extensions of this work are in progress with collaborators who have manipulated enhancers controlling IL-2Ra expression, with dramatic effects on autoimmune diabetes in mice. Imaging revealed changes in Treg expansion, micro-domain structure, and pSTAT5 status of Tconv vs. Treg cells that matched expectations from our new model of Treg function. These findings also illuminate how genetic control regions to which autoimmune susceptibility maps can allow escape of autoreactive cells into the effector pool by modestly changing expression of components of this intricate circuit we have characterized. To gain a deeper understanding of how T cells acquire their selective differentiated phenotypes or mediate effector function in the tumor micro-environment, we have begun development and application of a new correlative microscopy method. This entails 2P intravital imaging, the rapid fixation of the imaged tissue, and then multiplex 3D imaging. This permits linking the previous dynamic behavior of the T cells to their densely probed final phenotypic state. Progress has been made developing the various software tools required for image registration and the conduct of the multiplex analysis. With this tool, the current limitation of 2P IVM to 4-5 parameters can be overcome, because the characterization of cells in depth by IBEX multiplex imaging after the collection of dynamic data allows many different cell types to be tracked using a single color and then the identities deconvolved afterwards. To better understand the signaling events involved in T cell activation, the shaping of the effector and memory repertoire, and the function of T cells exposed in a chronic manner to antigen (e.g., chronic viral infections or cancer), we have developed a new approach that combines multiplex live reporters with single cell dynamic imaging in vitro. This allows us to manipulate the genotype of the T cells using CRISPR technology, while also monitoring the signaling response at multiple levels (p65 NFkB, ERK, p38, JNK, NFAT) in real time in response to TCR engagement by strong and weak ligands in the presence or absence of PD-1/PD-L1 and/or CD28/CD80 engagement. Our results have revealed a clear dichotomy in the triggering of certain signaling pathways by TCR, CD28, or the combination. The data also show that at all but saturating levels of strong agonist stimulation, PD-1 associated mechanisms affect signaling via the TCR (e.g., ERK or NFAT activation). Because TCR signaling via kinases is needed for CD28 signaling, this inhibition of TCR activity limits CD28 signaling, while SHP-2 that associates with PD-1 also can directly dephosphorylate any CD28 still modified by residual TCR signaling, amplifying the negative effects of PD-1 through combined blunting of signal 1 and signal 2. These findings have important implications for understanding how checkpoint blockade works and improving immunotherapies. This new system is also revealing the importance of catch bonds in effective TCR signaling and has proved useful in engineering TCR for enhanced anti-tumor activity with minimal risk of anti-self responses. The collection of molecular tools developed for this project have been deposited for use by other investigators. Lastly, a collaboration involving the use of DNA origami to simulate engagement of the TCR with high affinity ligands as is the case with CAR T cells had revealed the key effect of ligand density on the kinetics of MAPK activation, a phenomenon that fits well with earlier work from the LBS on the biochemistry of TCR signaling. Finally, we have begun a deep analysis of Tfh generation and germinal center (GC) function using our live and multiplex static 2D and 3D imaging platforms. Preliminary data suggests competition between T cells for a limiting number of antigen-activated B cells in maturing to the GC Tfh state, with implications for vaccine design in the sense that optimal matching of activated B cell frequency with activated T cell number may provide the system with more opportunities for somatic hypermutation to evolve an optimal humoral response. We are also testing various physical formations of antigens and various adjuvants, to examine how each affects the development of a humoral response. As part of this project and to support our broader imaging efforts, we are devoting substantial effort to development of new methods of quantitative analysis of spatial patterning of cells in complex tissues as revealed by multiplex imaging.