The immune synapses reveal aberrant functions of CD8 T cells during chronic HIV infection

It is well-established that chronic HIV infection causes persistent low-grade inflammation that induces premature aging of the immune system in HIV patient including senescence of memory and effector CD8 T cells. To uncover the reasons of gradually diminished potency of CD8 T cells from chronically HIV infected people, we have analyzed cellular morphology and dynamics of the synaptic interface followed exposure of peripheral polyclonal CD8 T cells at various differentiation stages to planar lipid bilayers. The above parameters were linked to pattern of degranulation that determines efficiency of CD8 T cells cytolytic response. We found a large fraction of naive T cells from HIV infected people developing mature synapses and demonstrating focused degranulation, a signature of a differentiated T cells. Further differentiation of aberrant naive T cells leads to development of anomalous effector T cells undermining their capacity to control HIV and other viruses that could be contained otherwise.

Defining the dynamics of naive CD4 and CD8 T cells across the mouse lifespan

Naive CD4 and CD8 T cells are part of the foundation of adaptive immune responses, but multiple aspects of their behaviour remain elusive. Newly generated T cells continue to develop after they leave the thymus and their dynamics and 'rules of entry' into the mature naive population are challenging to define. The extents to which naive T cells' capacities to survive or self-renew change as they age are also unclear. Further, much of what we know about their behaviour derives from studies in adults, both mouse and human. We know much less about naive T cell dynamics early in life, during which the thymus is highly active and peripheral T cell populations are rapidly established. For example, it has been suggested that neonatal mice are lymphopenic; if so, does this environment impact the behaviour of the earliest thymic emigrants, for example through altered rates of division and loss? In this study we integrate data from multiple experimental systems to construct models of naive CD4 and CD8 T cell population dynamics across the entire mouse lifespan. We infer that both subsets progressively increase their capacity to persist through survival mechanisms rather than through self-renewal, and find that this very simple model of adaptation describes the population dynamics of naive CD4 T cells from birth into old age. In addition, we find that newly generated naive CD8 T cells are lost at an elevated rate for the first 3-4 weeks of life, which may derive from transiently increased recruitment into conventional and virtual memory populations. We find no evidence for elevated rates of division of naive CD4 or CD8 T cells early in life and indeed estimate that these cells divide extremely rarely. Markers of proliferation within peripheral naive T cells are instead inherited from division during thymic development. We also find no evidence for feedback regulation of rates of division or loss of naive T cells at any age in healthy mice, challenging the dogma that their numbers are homeostatically regulated. Our analyses show how confronting an array of mechanistic mathematical models with diverse datasets can move us closer to a complete, and remarkably simple, picture of naive CD4 and CD8 T cell dynamics in mice.

Tonic IKK signalling regulates naive T cell survival in vivo by both repressing RIPK1 dependent extrinsic cell death pathways and independent activation of NF-κB

The Inhibitor of Kappa B Kinase (IKK) complex is a critical regulator of NF-κB activation. In addition, IKK has recently been shown to repress RIPK1 dependent extrinsic cell death pathways by directly phosphorylating RIPK1. Our previous work shows that normal thymopoiesis relies on IKK exclusively for repression of TNF triggered cell death pathways, and that NF-κB activation by IKK is redundant for development. The role of these pathways in mature naive T cells has not previously been reported. Here, we show that, like thymocytes, naive peripheral T cells require continued IKK1/2 expression for survival. In contrast, however, cell loss is only partially prevented by blocking extrinsic cell death pathways by either deleting Casp8 or inhibiting RIPK1 kinase activity. Inducible deletion of Rela in mature CD4+ T cells also results in a significant loss of naive CD4+ T cells and loss of IL7R expression, revealing an additional reliance upon NF-κB for long term survival of mature T cells. Together, these data show that IKK dependent survival of naive T cells depends upon both repression of extrinsic cell death pathways and activation of NF-κB survival programme.

Extracellular ATP Limits Homeostatic T Cell Migration Within Lymph Nodes

Whereas adenosine 5’-triphosphate (ATP) is the major energy source in cells, extracellular ATP (eATP) released from activated/damaged cells is widely thought to represent a potent damage-associated molecular pattern that promotes inflammatory responses. Here, we provide suggestive evidence that eATP is constitutively produced in the uninflamed lymph node (LN) paracortex by naïve T cells responding to C-C chemokine receptor type 7 (CCR7) ligand chemokines. Consistently, eATP was markedly reduced in naïve T cell-depleted LNs, including those of nude mice, CCR7-deficient mice, and mice subjected to the interruption of the afferent lymphatics in local LNs. Stimulation with a CCR7 ligand chemokine, CCL19, induced ATP release from LN cells, which inhibited CCR7-dependent lymphocyte migration in vitro by a mechanism dependent on the purinoreceptor P2X7 (P2X7R), and P2X7R inhibition enhanced T cell retention in LNs in vivo. These results collectively indicate that paracortical eATP is produced by naïve T cells in response to constitutively expressed chemokines, and that eATP negatively regulates CCR7-mediated lymphocyte migration within LNs via a specific subtype of ATP receptor, demonstrating its fine-tuning role in homeostatic cell migration within LNs.

Induction of memory-like CD8 + T cells and CD4 + T cells from human naïve T cells in culture

Memory T cells are crucial players in vertebrate adaptive immunity but their development is incompletely understood. Here we describe a method to produce human memory-like T cells from naïve human T cells in culture. Using commercially available human T cell differentiation kits, both purified naïve CD8 + T cells and purified naïve CD4 + T cells were activated via T cell receptor signaling and appropriate cytokines for several days in culture. All the T cell activators were then removed from the medium and the cultures were continued in hypoxic condition (1% O2 atmosphere) for several more days; during this period, most of the cells died, but some survived in a quiescent state for a month. The survivors had small round cell bodies, expressed differentiation markers characteristic of memory T cells and restarted proliferation when the T cell activators were added back. We could also induce memory-like T cells from naïve human T cells without hypoxia, if we froze the activated T cells or prepared the naïve T cells from chilled filter buffy coats.

Comparison of intestinal APC subsets from homeostatic and immunostimulatory environments

<p>Antigen presenting cells (APC) including dendritic cells (DC) play a key role in the initiation and direction of adaptive immune responses. Acting as sentinels in the tissue, DC sample antigen and traffic to the local lymph node where they present antigen to naïve T cells. The signals DC provide to naïve T cells determines the functional fate of the T cell and therefore, the type of immune response generated. At mucosal sites, such as the intestine, immune responses need to be carefully regulated due to the high antigenic load. For this reason, intestinal immune cells are highly specialised to prevent immune activation to innocuous antigens while still holding the capacity to induce potent responses to pathogenic microbes and helminths. Oral administration of antigen is associated with tolerance and the generation of FoxP3+ regulatory T cells (Tregs). Specialised lamina propria (LP) resident APC are required for the initiation of Treg differentiation in the mesenteric lymph nodes (MLN) through production of chemical mediators such as retinoic acid (RA). Ablation of these populations or restricted trafficking prevents the development of Tregs in mouse models thus supporting the essential role of APC in maintaining intestinal homeostasis. During infection, APC promote the induction of adaptive immune responses which neutralise threats. However, the APC subsets involved in this are not well defined. Pathologies such as food allergy and inflammatory bowel disease are thought to arise due to the development of aberrant immune responses. Food allergy can be modelled in mice using the mucosal adjuvant cholera toxin (CT) which has been shown to drive immunity to co-delivered antigens and is associated with the generation of IL-4 producing T helper 2 cells. Understanding the APC subsets involved in the initiation of intestinal immune responses could help in the development of targeted therapies for inflammatory bowel conditions. In this thesis, I show that oral administration of CT is followed by the appearance of a novel phenotype of DC in the intestinal LP and MLN. These DC differ functionally from DC at steady-state and may contribute to the generation of IL-4 producing T cells observed in the LP, MLN and spleen following oral administration of CT.</p>

Comparison of intestinal APC subsets from homeostatic and immunostimulatory environments

<p>Antigen presenting cells (APC) including dendritic cells (DC) play a key role in the initiation and direction of adaptive immune responses. Acting as sentinels in the tissue, DC sample antigen and traffic to the local lymph node where they present antigen to naïve T cells. The signals DC provide to naïve T cells determines the functional fate of the T cell and therefore, the type of immune response generated. At mucosal sites, such as the intestine, immune responses need to be carefully regulated due to the high antigenic load. For this reason, intestinal immune cells are highly specialised to prevent immune activation to innocuous antigens while still holding the capacity to induce potent responses to pathogenic microbes and helminths. Oral administration of antigen is associated with tolerance and the generation of FoxP3+ regulatory T cells (Tregs). Specialised lamina propria (LP) resident APC are required for the initiation of Treg differentiation in the mesenteric lymph nodes (MLN) through production of chemical mediators such as retinoic acid (RA). Ablation of these populations or restricted trafficking prevents the development of Tregs in mouse models thus supporting the essential role of APC in maintaining intestinal homeostasis. During infection, APC promote the induction of adaptive immune responses which neutralise threats. However, the APC subsets involved in this are not well defined. Pathologies such as food allergy and inflammatory bowel disease are thought to arise due to the development of aberrant immune responses. Food allergy can be modelled in mice using the mucosal adjuvant cholera toxin (CT) which has been shown to drive immunity to co-delivered antigens and is associated with the generation of IL-4 producing T helper 2 cells. Understanding the APC subsets involved in the initiation of intestinal immune responses could help in the development of targeted therapies for inflammatory bowel conditions. In this thesis, I show that oral administration of CT is followed by the appearance of a novel phenotype of DC in the intestinal LP and MLN. These DC differ functionally from DC at steady-state and may contribute to the generation of IL-4 producing T cells observed in the LP, MLN and spleen following oral administration of CT.</p>