Functional Biology Of T Cells

National Institute Of Allergy And Infectious Diseases

Investigators

Linked publications, trials & patents

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 cell 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. Over the past several years we have used our advanced imaging platforms to determine the fate of auto-activated CD4 T cells controlled by Treg. A highly quantitative spatial statistical analysis revealed that Tregs are concentrated in micro-domains around self-responsive 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 (IL-2Ra) 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. 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. We have now collaborated with investigators at UCSF who have manipulated enhancers controlling IL-2Ra expression selectively in Tconv vs. Treg T cells. Remarkably, minor changes in the rate of upregulation of IL-2Ra after T cell stimulation have dramatic effects on autoimmune diabetes in mice. Slightly delaying Treg CD25 expression causes NOD mice to develop diabetes in a more rapid and more penetrant fashion, whereas slightly delaying CD25 upregulation by Tconv cells yields animals completely resistant to diabetes even after treatment with anti-PD-1 antibody. Imaging matched expectations from our computational model of Treg function once adjustments were made for the NOD background. These findings 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. 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 of a new correlative microscopy method (see also AI000545-35). This entails 2P intravital imaging, the rapid fixation of the imaged tissue, and then multiplex 3D imaging, linking dynamic behavior of the T cells to their 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 approach, 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 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 method that combines multiplex live reporters with single cell dynamic imaging in vitro. This method, called FILMSTAR, 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 pMHC ligands in the presence or absence of PD-1/PD-L1 and/or CD28/CD80 engagement. Our results show a clear dichotomy in the triggering of certain signaling pathways by TCR, CD28, or the combination. The data also show that PD-1 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 phosphatases that associate 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, has proved useful in engineering TCR for enhanced anti-tumor activity with minimal risk of anti-self responses, and confirmed reports of cis- interaction of CD80 and PD-L1. The latter finding amplifies the linked behavior of TCR and CD28 signaling in that as an antigen presenting cell increases CD80 expression, it inactivates the pool of PD-L1 with respect to PD-1 binding. 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, with a specific emphasis on the molecular events and tissue structures involved in the generation and support of long-lived plasma cells. These cells are critical for sustained protection by antibodies after vaccination and how best to develop such long-lived responses remains a major goal of vaccine research. 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. Lastly, we are using our IBEX methods and Ce3D-IBEX to create detailed atlases of the human and mouse thymus across developmental stages. In a collaboration with the Mathis laboratory at Harvard, we are specifically analyzing the localization, cell-cell interactions, and function of the many mimetic cells believed to play a critical role in functional self-tolerance.