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 or tumors to act to constrain malignant cell growth. They can also cause autoimmune pathology. Most activated T-cells die, but some remain as memory cells. 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 and influence the development of the long-lived plasma cells that are responsible for maintaining protective antibodies levels following infection or vaccination. This project attempts to gain both a qualitative and quantitative understanding of the activation, differentiation, migration, cell-cell interaction, 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. It also investigates how T cell activity affects durable antibody responses, the niches involved in supporting the plasma cells that give rise to these antibodies, and what methods will enable assessment of lymphocyte function in ethically accessible human tissues. 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 and activation of the 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 enter cell cycle, but without access to IL-2 they rapidly die, pruning the repertoire of these autoreactive cells. A mathematical model showed 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. Collaboration with scientists at Univ. Chicago have expanded our understanding of how small changes in the parameters contained in our computational model of Treg function affect autoimmunity. Under homeostatic conditions, Tregs do not need to recognize the same antigen as the Tconv cells they regulate. This is also true even in the face of generalized inflammation. However, if inflammation is accompanied by an increase in self-antigen presentation, prevention of autoimmune disease requires matching the Treg specificity with that of the cells they need to control. These findings correspond well with the expectations of our computer model. By competing for antigen recognition, the specificity matched Tregs reduce TCR input, keeping IL-2 production within the range that can be consumed by the Tregs. These data have important implications for understanding how tissue damage and/or infection can instigate autoimmune responses and these findings were published in the journal Science. 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 nearly completed development of a new correlative microscopy method (see also AI000545-37). 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. The various software tools required for image registration, the conduct of the multiplex analysis, and cell tracking are now being deployed for experiments in multiple immunization and other models. 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 developed a method that combined 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 showed that PD-1 inhibits signaling via the TCR (e.g., ERK or NFAT activation). These findings have important implications for understanding how checkpoint blockade works, have 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 have served as the basis for new studies of T-cell dependent antibody responses as detailed below. 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. We are testing various physical formations of antigens and various adjuvants with a focus on those in use in humans in licensed vaccines, 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 and have now developed a 30-plex panel for deep examination of the spatial organization of the bone marrow, with special emphasis on the cellular niches within which plasma cells reside. Very recently, these imaging studies have revealed an unexpected synaptic association of plasma cells with another immune cell type and experiments are ongoing to better understand the significance and function of this novel interaction – our initial hypothesis is that this interaction is involved in eliminating plasma cells unable to properly buffer the ER stress of their high protein synthetic rate when producing antibodies, with such pruning critical to avoiding development of plasma cell malignancies (multiple myeloma) due to genotoxic effects of the stress response. We are also examining the regulation of CD80 vs. PD-L1 expression by germinal center B cells. This past year we have developed a mouse with a mutation in CD80 that eliminates its capacity for cis-interaction with PD-L1 and are now examining these mice after immunization to see if this mutation decreases affinity maturation as we predict. Lastly, we are using our IBEX methods and Ce3D-IBEX to create detailed atlases of the human and mouse thymus across developmental stages. Our studies of the human thymus have revealed new insights into the localization of various epithelial cell subsets and the differential timing of movement of maturing CD4 vs. CD8 T cells from cortex to medulla. In a collaboration with the Mathis laboratory at Harvard, we have developed a 60 plex panel for the study of the mouse thymus and especially ‘mimetic cells’ involved in central tolerance to peripheral tissue antigens., along with a smaller panel we have deployed using our Ce3D-IBEX method for 3D tissue imaging that reveals an intriguing patterning of the mimetic cells and allows mapping these cells relative to blood vessels and nerves that are otherwise not easily charted in thin 2D sections.