Chemokine-mediated control of immunity to tumours and infectious pathogens

The ability of immune cells to migrate to distinct niches and peripheral sites is critical for their appropriate differentiation and for execution of their effector functions. This migration is facilitated to a large degree by the expression of chemokine receptors, which allow for migration in a spatiotemporally-controlled manner. The work presented in this thesis addresses two distinct issues regarding how regulation of immune cell migration affects development of anti-tumour immunity and infectious immunity. In the first part of this thesis, a novel role for the atypical chemokine receptor ACKR4 in controlling anti-tumour immune responses was identified. As a scavenging receptor, ACKR4 regulates the bioavailability of the CCR7 ligands, CCL19 and CCL21, and the CCR9 ligand, CCL25. These ligands have previously been shown to be critical for many aspects of immune homeostasis, as well as contributing to tumour cell growth and metastasis. However, the contribution of ACKR4 in regulating tumour-specific responses has been unclear. Using multiple orthotopic, transgenic and chemically-induced models of cancer, loss of ACKR4 resulted in inhibited tumour growth. In the absence of ACKR4, enhanced CCL21 levels were associated with enhanced tumour infiltration of IFNy+ CD8+ T cells. The reduced tumour growth seen was dependent on the enhanced CD8+ response, with depletion of CD8+ T cells restoring growth of Ackr4–/– tumours to wildtype levels. The enhanced CD8+ T cell response was not a result of altered priming in draining lymph nodes, although there was increased intratumoural proliferation of CD8+ T cells. Furthermore, ACKR4-deficient tumours showed increased retention of CD103+ DCs, with these cells previously being shown to be critical for effective recruitment of CD8+ T cells to tumours. Moreover, intratumoural administration of CCL21 into wildtype tumours also enhanced the accumulation of DCs, suggesting a direct role for the scavenging ability of ACKR4. These data support the notion that ACKR4, through its regulation of CCL21 bioavailability, controls DC migration in tumours thus regulating the development of anti-tumour immune responses. Furthermore, multiple immunotherapies show increased efficacy in the absence of ACKR4, suggesting ACKR4 may be useful as a potential novel target for immunotherapy. In the second part of this thesis, the role of CCR2 on memory CD4+ T cells was explored. Relatively little is understood about the generation, maintenance and effector functions of memory CD4+ T cells, despite correlations with improved disease outcomes. Furthermore, how these cells migrate to inflammatory sites is still largely unknown. In this project, CCR2 was identified as being enriched on antigen-specific memory CD4+ T cells in response to infection with the extracellular bacteria Streptococcus pneumoniae and infection with influenza A virus. Competitive co-transfer of wildtype and CCR2- deficient TCR-transgenic CD4+ T cells showed enhanced contraction of Ccr2–/– cells, suggesting a cell-intrinsic role for CCR2 in CD4+ T cell maintenance. CCR2-deficient effector cells were unaffected in their ability to secrete cytokines or enter into effector sites. Moreover, despite being numerically reduced at memory timepoints compared with CCR2-sufficent cells, they were equally capable of expanding upon secondary challenge. These data highlight CCR2 as an important regulator of CD4+ T cell memory maintenance. Taken together, this project has furthered our understanding of the complexity of cell migration in dictating immune responses. The identification of CCR2 as a mediator of memory CD4+ T cell generation may allow further investigation into how these cells are induced and maintained. In ACKR4, a novel level of post-transcriptional regulation of intratumoural DC trafficking has been identified, with this having the potential to be a tractable target for therapeutic manipulation in malignant disease.Thesis (Ph.D.) -- University of Adelaide, School of Biological Sciences, 201

AutoSpill is a principled framework that simplifies the analysis of multichromatic flow cytometry data.

Compensating in flow cytometry is an unavoidable challenge in the data analysis of fluorescence-based flow cytometry. Even the advent of spectral cytometry cannot circumvent the spillover problem, with spectral unmixing an intrinsic part of such systems. The calculation of spillover coefficients from single-color controls has remained essentially unchanged since its inception, and is increasingly limited in its ability to deal with high-parameter flow cytometry. Here, we present AutoSpill, an alternative method for calculating spillover coefficients. The approach combines automated gating of cells, calculation of an initial spillover matrix based on robust linear regression, and iterative refinement to reduce error. Moreover, autofluorescence can be compensated out, by processing it as an endogenous dye in an unstained control. AutoSpill uses single-color controls and is compatible with common flow cytometry software. AutoSpill allows simpler and more robust workflows, while reducing the magnitude of compensation errors in high-parameter flow cytometry

Impaired HA-specific T follicular helper cell and antibody responses to influenza vaccination are linked to inflammation in humans.

Funder: National Institute for Health Research (NIHR)Antibody production following vaccination can provide protective immunity to subsequent infection by pathogens such as influenza viruses. However, circumstances where antibody formation is impaired after vaccination, such as in older people, require us to better understand the cellular and molecular mechanisms that underpin successful vaccination in order to improve vaccine design for at-risk groups. Here, by studying the breadth of anti-haemagglutinin (HA) IgG, serum cytokines, and B and T cell responses by flow cytometry before and after influenza vaccination, we show that formation of circulating T follicular helper (cTfh) cells was associated with high-titre antibody responses. Using Major Histocompatability Complex (MHC) class II tetramers, we demonstrate that HA-specific cTfh cells can derive from pre-existing memory CD4+ T cells and have a diverse T cell receptor (TCR) repertoire. In older people, the differentiation of HA-specific cells into cTfh cells was impaired. This age-dependent defect in cTfh cell formation was not due to a contraction of the TCR repertoire, but rather was linked with an increased inflammatory gene signature in cTfh cells. Together, this suggests that strategies that temporarily dampen inflammation at the time of vaccination may be a viable strategy to boost optimal antibody generation upon immunisation of older people

Increasing frailty is associated with higher prevalence and reduced recognition of delirium in older hospitalised inpatients: results of a multi-centre study

Purpose: Delirium is a neuropsychiatric disorder delineated by an acute change in cognition, attention, and consciousness. It is common, particularly in older adults, but poorly recognised. Frailty is the accumulation of deficits conferring an increased risk of adverse outcomes. We set out to determine how severity of frailty, as measured using the CFS, affected delirium rates, and recognition in hospitalised older people in the United Kingdom. Methods: Adults over 65 years were included in an observational multi-centre audit across UK hospitals, two prospective rounds, and one retrospective note review. Clinical Frailty Scale (CFS), delirium status, and 30-day outcomes were recorded. Results: The overall prevalence of delirium was 16.3% (483). Patients with delirium were more frail than patients without delirium (median CFS 6 vs 4). The risk of delirium was greater with increasing frailty [OR 2.9 (1.8–4.6) in CFS 4 vs 1–3; OR 12.4 (6.2–24.5) in CFS 8 vs 1–3]. Higher CFS was associated with reduced recognition of delirium (OR of 0.7 (0.3–1.9) in CFS 4 compared to 0.2 (0.1–0.7) in CFS 8). These risks were both independent of age and dementia. Conclusion: We have demonstrated an incremental increase in risk of delirium with increasing frailty. This has important clinical implications, suggesting that frailty may provide a more nuanced measure of vulnerability to delirium and poor outcomes. However, the most frail patients are least likely to have their delirium diagnosed and there is a significant lack of research into the underlying pathophysiology of both of these common geriatric syndromes

Context-dependent effects of IL-2 rewire immunity into distinct cellular circuits.

Funder: Fondation pour l'Aide à la Recherche sur la Sclérose en PlaquesFunder: Alzheimer's AssociationFunder: European Research CouncilFunder: Fondation pour l’Aide à la Recherche sur la Sclérose en PlaquesFunder: VetenskapsrådetFunder: Alzheimer’s AssociationFunder: Fonds Wetenschappelijk OnderzoekFunder: Vlaams Instituut voor BiotechnologieInterleukin 2 (IL-2) is a key homeostatic cytokine, with therapeutic applications in both immunogenic and tolerogenic immune modulation. Clinical use has been hampered by pleiotropic functionality and widespread receptor expression, with unexpected adverse events. Here, we developed a novel mouse strain to divert IL-2 production, allowing identification of contextual outcomes. Network analysis identified priority access for Tregs and a competitive fitness cost of IL-2 production among both Tregs and conventional CD4 T cells. CD8 T and NK cells, by contrast, exhibited a preference for autocrine IL-2 production. IL-2 sourced from dendritic cells amplified Tregs, whereas IL-2 produced by B cells induced two context-dependent circuits: dramatic expansion of CD8+ Tregs and ILC2 cells, the latter driving a downstream, IL-5-mediated, eosinophilic circuit. The source-specific effects demonstrate the contextual influence of IL-2 function and potentially explain adverse effects observed during clinical trials. Targeted IL-2 production therefore has the potential to amplify or quench particular circuits in the IL-2 network, based on clinical desirability

A distal enhancer at risk locus 11q13.5 promotes suppression of colitis by T

Genetic variations underlying susceptibility to complex autoimmune and allergic diseases are concentrated within noncoding regulatory elements termed enhancers1. The functions of a large majority of disease-associated enhancers are unknown, in part owing to their distance from the genes they regulate, a lack of understanding of the cell types in which they operate, and our inability to recapitulate the biology of immune diseases in vitro. Here, using shared synteny to guide loss-of-function analysis of homologues of human enhancers in mice, we show that the prominent autoimmune and allergic disease risk locus at chromosome 11q13.52-7 contains a distal enhancer that is functional in CD4+ regulatory T (Treg) cells and required for Treg-mediated suppression of colitis. The enhancer recruits the transcription factors STAT5 and NF-κB to mediate signal-driven expression of Lrrc32, which encodes the protein glycoprotein A repetitions predominant (GARP). Whereas disruption of the Lrrc32 gene results in early lethality, mice lacking the enhancer are viable but lack GARP expression in Foxp3+ Treg cells, which are unable to control colitis in a cell-transfer model of the disease. In human Treg cells, the enhancer forms conformational interactions with the promoter of LRRC32 and enhancer risk variants are associated with reduced histone acetylation and GARP expression. Finally, functional fine-mapping of 11q13.5 using CRISPR-activation (CRISPRa) identifies a CRISPRa-responsive element in the vicinity of risk variant rs11236797 capable of driving GARP expression. These findings provide a mechanistic basis for association of the 11q13.5 risk locus with immune-mediated diseases and identify GARP as a potential target in their therapy

Astrocyte-targeted gene delivery of interleukin 2 specifically increases brain-resident regulatory T cell numbers and protects against pathological neuroinflammation

International audienceThe ability of immune-modulating biologics to prevent and reverse pathology has transformed recent clinical practice. Full utility in the neuroinflammation space, however, requires identification of both effective targets for local immune modulation and a delivery system capable of crossing the blood–brain barrier. The recent identification and characterization of a small population of regulatory T (T reg ) cells resident in the brain presents one such potential therapeutic target. Here, we identified brain interleukin 2 (IL-2) levels as a limiting factor for brain-resident T reg cells. We developed a gene-delivery approach for astrocytes, with a small-molecule on-switch to allow temporal control, and enhanced production in reactive astrocytes to spatially direct delivery to inflammatory sites. Mice with brain-specific IL-2 delivery were protected in traumatic brain injury, stroke and multiple sclerosis models, without impacting the peripheral immune system. These results validate brain-specific IL-2 gene delivery as effective protection against neuroinflammation, and provide a versatile platform for delivery of diverse biologics to neuroinflammatory patients

Astrocyte-targeted gene delivery of interleukin 2 specifically increases brain-resident regulatory T cell numbers and protects against pathological neuroinflammation.

The ability of immune-modulating biologics to prevent and reverse pathology has transformed recent clinical practice. Full utility in the neuroinflammation space, however, requires identification of both effective targets for local immune modulation and a delivery system capable of crossing the blood-brain barrier. The recent identification and characterization of a small population of regulatory T (Treg) cells resident in the brain presents one such potential therapeutic target. Here, we identified brain interleukin 2 (IL-2) levels as a limiting factor for brain-resident Treg cells. We developed a gene-delivery approach for astrocytes, with a small-molecule on-switch to allow temporal control, and enhanced production in reactive astrocytes to spatially direct delivery to inflammatory sites. Mice with brain-specific IL-2 delivery were protected in traumatic brain injury, stroke and multiple sclerosis models, without impacting the peripheral immune system. These results validate brain-specific IL-2 gene delivery as effective protection against neuroinflammation, and provide a versatile platform for delivery of diverse biologics to neuroinflammatory patients