Formation, Positioning, Motility, and Function of Tissue Resident Memory CD8+ T cells after Influenza Infection

Rationale  

Tissue resident memory T cells (TRM) are non-recirculating CD8+ T cells that become established in peripheral tissues after an infection. Upon re-encounter with the same or related pathogen(s), these memory T cells rapidly reactivate and provide immediate effector function that can mean the difference between life and death in a lethal challenge model.  They tend to be specific for conserved antigens, and in the case of influenza, could be part of the solution to achieving more broadly cross-reactive and universal immunity. Understanding how they are regulated, how they mediate optimal protection, and how they are established and maintained are critical goals of this project. Besides the markers used to identify the T cell types (CD3, CD8, CD44, CD62L, CD69), several other cell surface markers are commonly used to identify memory T cell subsets in the tissues.  CD49a, when paired to integrin beta-1 to form VLA-1, is a receptor for collagen in the extracellular matrix and is the prototypic TRM marker first used to define these cells in the tissue by us in 2004. Blockade or deletion of CD49a leads to loss of TRM in the periphery and loss of heterosubtypic immune protection. CD103, when paired with beta-7 integrin, binds to E-cadherin expressed in the junctions between epithelial cells. CD103 has been more commonly used than CD49a to mark TRM, but as we will show, only identifies a subset of these cells giving an incomplete picture. The majority of TRM express CD49a, with about 30% of those co-expressing CD103. Using CD49a and CD103, four subsets of memory CD8 T cells can be identified in the lung. Each has distinct gene expression and effector functions. Markers are all well and good, but do not tell us what their function is. Little has been done to determine the functions of CD49a and CD103 besides some deletion or inhibition studies followed by a census of the cells remaining in the tissues. Our research revealed that CD49a is needed for locomotion of TRM in the tissue, while CD103 acts to tether the cells to  epithelial cell junctions.  We are testing hypotheses related to how each of these adhesion molecules acts to position memory T cells in different anatomical locations, regulate communication with the epithelium, are associated with genetic programming linked to functional differentiation, thereby regulating CD8+ T cell motility, survival, and optimal immune protection.

Aim  1

Determine the mechanisms that determine differentiation, establishment, and maintenance of TRM subsets after influenza infection. We have used flow cytometry of the integrins CD49a and CD103 to define four different subsets of memory CD8 T cells found in the airways and lung tissue. RNAseq data shows tight clustering of each of these subsets, suggesting there are transcriptional and epigenetic changes that differentiate them. Knowing how TRM subsets are genetically regulated may lead to strategies to enhance their generation and maintenance, which could improve cross-protective immunity to influenza. In this Aim we will test the hypothesis that signals from the airway and lung environments during influenza infection drive permanent changes in gene expression partially mediated through specific transcription factors and epigenetic modifications.

Aim 2 

Investigate mechanisms of T cell-epithelial cell-matrix interactions required for motility and positioning in the airways. Sensing changes in the tissue microenvironment and responding to cues are critical for memory T cells to provide immune surveillance and protection. Integrins and other adhesion molecules mediate T cell interaction and communication with the environment and their ability to migrate and position optimally in the tissue. Our preliminary data show that CD49a supports locomotion on collagen and is critical for the maintenance of TRM.   CD103 supports tethering to E-cadherin expressed by epithelial cells and is important for the accumulation of CD8 T cells in the airways. Imaging has also revealed close association of OT-I T cells with adjacent epithelial cells, and novel preliminary RNAseq data has identified expression of three tight junction genes by the CD49a+ CD103+ TRM. Together, the hypothesis is formed that CD8 TRM motility and surveillance in the epithelium involves active formation of junctions with epithelial cells via CD103/E-cadherin and other junctional proteins, and locomotion on collagen. These interactions are expected to specifically position the cells in the epithelium and regulate immune surveillance.

Aim 3

Determine the functions of CD49a and CD103 in optimizing immune protection. Preliminary data demonstrates that TRM from the respiratory tract can be divided into four subsets based on integrin expression, and we see further functional differences in each of the four subsets, with differential effector cytokine secretion and cytotoxic potential. RNAseq data provides evidence for expression of junctional proteins, potentially providing other mechanisms that regulate CD8 TRM motility and positioning. This aim seeks to test whether the integrins CD49a and CD103 act in concert with junctional proteins to provide optimal immune surveillance and effector function upon secondary encounter with pathogen. In this Aim we seek to test this hypothesis through experimental interventions that perturb EC/T cell interactions, followed by secondary virus infection.