CXCR2 inhibition enables NASH-HCC immunotherapy.

OBJECTIVE: Hepatocellular carcinoma (HCC) is increasingly associated with non-alcoholic steatohepatitis (NASH). HCC immunotherapy offers great promise; however, recent data suggests NASH-HCC may be less sensitive to conventional immune checkpoint inhibition (ICI). We hypothesised that targeting neutrophils using a CXCR2 small molecule inhibitor may sensitise NASH-HCC to ICI therapy. DESIGN: Neutrophil infiltration was characterised in human HCC and mouse models of HCC. Late-stage intervention with anti-PD1 and/or a CXCR2 inhibitor was performed in murine models of NASH-HCC. The tumour immune microenvironment was characterised by imaging mass cytometry, RNA-seq and flow cytometry. RESULTS: Neutrophils expressing CXCR2, a receptor crucial to neutrophil recruitment in acute-injury, are highly represented in human NASH-HCC. In models of NASH-HCC lacking response to ICI, the combination of a CXCR2 antagonist with anti-PD1 suppressed tumour burden and extended survival. Combination therapy increased intratumoural XCR1+ dendritic cell activation and CD8+ T cell numbers which are associated with anti-tumoural immunity, this was confirmed by loss of therapeutic effect on genetic impairment of myeloid cell recruitment, neutralisation of the XCR1-ligand XCL1 or depletion of CD8+ T cells. Therapeutic benefit was accompanied by an unexpected increase in tumour-associated neutrophils (TANs) which switched from a protumour to anti-tumour progenitor-like neutrophil phenotype. Reprogrammed TANs were found in direct contact with CD8+ T cells in clusters that were enriched for the cytotoxic anti-tumoural protease granzyme B. Neutrophil reprogramming was not observed in the circulation indicative of the combination therapy selectively influencing TANs. CONCLUSION: CXCR2-inhibition induces reprogramming of the tumour immune microenvironment that promotes ICI in NASH-HCC.

Neutrophil dynamics in the tumor microenvironment.

The tumor microenvironment profoundly influences the behavior of recruited leukocytes and tissue-resident immune cells. These immune cells, which inherently have environmentally driven plasticity necessary for their roles in tissue homeostasis, dynamically interact with tumor cells and the tumor stroma and play critical roles in determining the course of disease. Among these immune cells, neutrophils were once considered much more static within the tumor microenvironment; however, some of these earlier assumptions were the product of the notorious difficulty in manipulating neutrophils in vitro. Technological advances that allow us to study neutrophils in context are now revealing the true roles of neutrophils in the tumor microenvironment. Here we discuss recent data generated by some of these tools and how these data might be synthesized into more elegant ways of targeting these powerful and abundant effector immune cells in the clinic.

Expression of the B cell differentiation factor BAFF and chemokine CXCL13 in a murine model of Respiratory Syncytial Virus infection.

Innate immune responses are known to influence the subsequent development of adaptive immunity. We have previously shown that RSV infection of human airway epithelial cells results in production of the B cell growth factor, BAFF. To better understand how the airway responds to RSV infection by production of this and other factors to support or enhance local B cell responses to infection, we analysed the lung expression of BAFF and B cell homeostatic chemokines CXCL12, CXCL13, CCL19 and CCL21 in a murine model of RSV infection. Following infection with A2 strain RSV, the highest RSV N gene expression was observed at day 4 after challenge with virus. In contrast, two peaks of elevated BAFF expression at days 2 and 7 were observed. CXCL13 was significantly elevated at days 1, 2 and 7. CXCL12, CCL19 and CCL21 were expressed within lung tissue from control and RSV challenged animals but no significant difference in expression was found. Immunofluorescence showed BAFF to be present throughout the tissue however CXCL13 expression was localized to cell rich areas probably constituting lymphoid aggregates. Our results define the kinetics of B cell chemoattractant and growth factor expression during RSV infection and indicate an important role for these cytokines in the airway response to RSV infection.

Enteric helminth-induced type I interferon signaling protects against pulmonary virus infection through interaction with the microbiota.

BACKGROUND: Helminth parasites have been reported to have beneficial immunomodulatory effects in patients with allergic and autoimmune conditions and detrimental consequences in patients with tuberculosis and some viral infections. Their role in coinfection with respiratory viruses is not clear. OBJECTIVE: Here we investigated the effects of strictly enteric helminth infection with Heligmosomoides polygyrus on respiratory syncytial virus (RSV) infection in a mouse model. METHODS: A murine helminth/RSV coinfection model was developed. Mice were infected by means of oral gavage with 200 stage 3 H polygyrus larvae. Ten days later, mice were infected intranasally with either RSV or UV-inactivated RSV. RESULTS: H polygyrus-infected mice showed significantly less disease and pulmonary inflammation after RSV infection associated with reduced viral load. Adaptive immune responses, including TH2 responses, were not essential because protection against RSV was maintained in Rag1-/- and Il4rα-/- mice. Importantly, H polygyrus infection upregulated expression of type I interferons and interferon-stimulated genes in both the duodenum and lung, and its protective effects were lost in both Ifnar1-/- and germ-free mice, revealing essential roles for type I interferon signaling and microbiota in H polygyrus-induced protection against RSV. CONCLUSION: These data demonstrate that a strictly enteric helminth infection can have remote protective antiviral effects in the lung through induction of a microbiota-dependent type I interferon response.

Cathelicidins Have Direct Antiviral Activity against Respiratory Syncytial Virus In Vitro and Protective Function In Vivo in Mice and Humans.

Respiratory syncytial virus (RSV) is a leading cause of respiratory tract infection in infants, causing significant morbidity and mortality. No vaccine or specific, effective treatment is currently available. A more complete understanding of the key components of effective host response to RSV and novel preventative and therapeutic interventions are urgently required. Cathelicidins are host defense peptides, expressed in the inflamed lung, with key microbicidal and modulatory roles in innate host defense against infection. In this article, we demonstrate that the human cathelicidin LL-37 mediates an antiviral effect on RSV by inducing direct damage to the viral envelope, disrupting viral particles and decreasing virus binding to, and infection of, human epithelial cells in vitro. In addition, exogenously applied LL-37 is protective against RSV-mediated disease in vivo, in a murine model of pulmonary RSV infection, demonstrating maximal efficacy when applied concomitantly with virus. Furthermore, endogenous murine cathelicidin, induced by infection, has a fundamental role in protection against disease in vivo postinfection with RSV. Finally, higher nasal levels of LL-37 are associated with protection in a healthy human adult RSV infection model. These data lead us to propose that cathelicidins are a key, nonredundant component of host defense against pulmonary infection with RSV, functioning as a first point of contact antiviral shield and having additional later-phase roles in minimizing the severity of disease outcome. Consequently, cathelicidins represent an inducible target for preventative strategies against RSV infection and may inform the design of novel therapeutic analogs for use in established infection.

Effector and central memory T helper 2 cells respond differently to peptide immunotherapy.

Peptide immunotherapy (PIT) offers realistic prospects for the treatment of allergic diseases, including allergic asthma. Much is understood of the behavior of naive T cells in response to PIT. However, treatment of patients with ongoing allergic disease requires detailed understanding of the responses of allergen-experienced T cells. CD62L expression by allergen-experienced T cells corresponds to effector/effector memory (CD62L(lo)) and central memory (CD62L(hi)) subsets, which vary with allergen exposure (e.g., during, or out with, pollen season). The efficacy of PIT on different T helper 2 (Th2) cell memory populations is unknown. We developed a murine model of PIT in allergic airway inflammation (AAI) driven by adoptively transferred, traceable ovalbumin-experienced Th2 cells. PIT effectively suppressed AAI driven by unfractionated Th2 cells. Selective transfer of CD62L(hi) and CD62L(lo) Th2 cells revealed that these two populations behaved differently from one another and from previously characterized (early deletional) responses of naive CD4(+) T cells to PIT. Most notably, allergen-reactive CD62L(lo) Th2 cells were long-lived within the lung after PIT, before allergen challenge, in contrast to CD62L(hi) Th2 cells. Despite this, PIT was most potent against CD62L(lo) Th2 cells in protecting from AAI, impairing their ability to produce Th2 cytokines, whereas this capacity was heightened in PIT-treated CD62L(hi) Th2 cells. We conclude that Th2 cells do not undergo an early deletional form of tolerance after PIT. Moreover, memory Th2 subsets respond differently to PIT. These findings have implications for the clinical translation of PIT in different allergic scenarios.

Combination peptide immunotherapy based on T-cell epitope mapping reduces allergen-specific IgE and eosinophilia in allergic airway inflammation.

Peptide immunotherapy using soluble peptides containing allergen-derived immunodominant T-cell epitopes holds therapeutic promise for allergic asthma. Previous studies in BALB/c mice using the immunodominant peptide epitope of chicken ovalbumin (p323-339) have been unable to demonstrate therapeutic effects in ovalbumin-induced allergic airway inflammation. We have previously shown that intravenous application of p323-339 can effectively tolerise p323-339-reactive T cells in a non-allergic model in C57BL/6 mice. This study aimed to assess the effects of using p323-339 immunotherapy in a C57BL/6 model of ovalbumin-induced allergic airway inflammation, identify any additional epitopes recognized by the ovalbumin-responsive T-cell repertoire in C57BL/6 mice and assess the effects of combination peptide immunotherapy in this model. Ovalbumin-reactive T-cell lines were generated from ovalbumin-immunized C57BL/6 mice and proliferative responses to a panel of overlapping peptides covering the ovalbumin sequence were assessed. Soluble peptides (singly or combined) were administered intravenously to C57BL/6 mice before the induction of ovalbumin-induced allergic airway inflammation. Peptide immunotherapy using the 323-339 peptide alone did not reduce the severity of allergic airway inflammation. An additional immunodominant T-cell epitope in ovalbumin was identified within the 263-278 sequence. Combination peptide immunotherapy, using the 323-339 and 263-278 peptides together, reduced eosinophilia in the bronchoalveolar lavage and ovalbumin-specific IgE, with apparent reductions in interleukin-5 and interleukin-13. Characterization of the T-cell response to a model allergen has allowed the development of combination peptide immunotherapy with improved efficacy in allergic airway inflammation. This model holds important potential for future mechanistic studies using peptide immunotherapy in allergy.