Mechanisms of antibody mediated immunity - Distinct in early life.

Immune responses in early life are characterized by a failure to robustly generate long-lasting protective responses against many common pathogens or upon vaccination. This is associated with a reduced ability to generate T-cell dependent high affinity antibodies. This review highlights the differences in T-cell dependent antibody responses observed between infants and adults, in particular focussing on the alterations in immune cell function that lead to reduced T follicular helper cell-B cell crosstalk within germinal centres in early life. Understanding the distinct functional characteristics of early life humoral immunity, and how these are regulated, will be critical in guiding age-appropriate immunological interventions in the very young.

Role of epithelial barrier function in inducing type 2 immunity following early-life viral infection.

BACKGROUND: Preschool wheeze attacks triggered by recurrent viral infections, including respiratory syncytial virus (RSV), are associated with an increased risk of childhood asthma. However, mechanisms that lead to asthma following early-life viral wheezing remain uncertain. METHODS: To investigate a causal relationship between early-life RSV infections and onset of type 2 immunity, we developed a neonatal murine model of recurrent RSV infection, in vivo and in silico, and evaluated the dynamical changes of altered airway barrier function and downstream immune responses, including eosinophilia, mucus secretion and type 2 immunity. RESULTS: RSV infection of neonatal BALB/c mice at 5 and 15 days of age induced robust airway eosinophilia, increased pulmonary CD4+ IL-13+ and CD4+ IL-5+ cells, elevated levels of IL-13 and IL-5 and increased airway mucus at 20 days of age. Increased bronchoalveolar lavage albumin levels, suggesting epithelial barrier damage, were present and persisted following the second RSV infection. Computational in silico simulations demonstrated that recurrent RSV infection resulted in severe damage of the airway barrier (epithelium), triggering the onset of type 2 immunity. The in silico results also demonstrated that recurrent infection is not always necessary for the development of type 2 immunity, which could also be triggered with single infection of high viral load or when the epithelial barrier repair is compromised. CONCLUSIONS: The neonatal murine model demonstrated that recurrent RSV infection in early life alters airway barrier function and promotes type 2 immunity. A causal relationship between airway barrier function and type 2 immunity was suggested using in silico model simulations.

IL-6 and IL-27 play both distinct and redundant roles in regulating CD4 T-cell responses during chronic viral infection.

The IL-6 cytokine family signals through the common signal transduction molecule gp130 combined with a cytokine-specific receptor. Gp130 signaling on CD4 T cells is vital in controlling chronic infection of mice with lymphocytic choriomeningitis virus clone 13 (LCMV Cl13), but the precise role of individual members of the IL-6 cytokine family is not fully understood. Transcriptional analysis highlighted the importance of gp130 signaling in promoting key processes in CD4 T cells after LCMV Cl13 infection, particularly genes associated with T follicular helper (Tfh) cell differentiation and IL-21 production. Further, Il27r-/-Il6ra-/- mice failed to generate antibody or CD8 T-cell immunity and to control LCMV Cl13. Transcriptomics and phenotypic analyses of Il27r-/-Il6ra-/- Tfh cells revealed that IL-6R and IL-27R signaling was required to activate key pathways within CD4 T cells. IL-6 and IL-27 signaling has distinct and overlapping roles, with IL-6 regulating Tfh differentiation, IL-27 regulating CD4 T cell survival, and both redundantly promoting IL-21.

T helper 2 cells in asthma.

Allergic asthma is among the most common immune-mediated diseases across the world, and type 2 immune responses are thought to be central to pathogenesis. The importance of T helper 2 (Th2) cells as central regulators of type 2 responses in asthma has, however, become less clear with the discovery of other potent innate sources of type 2 cytokines and innate mediators of inflammation such as the alarmins. This review provides an update of our current understanding of Th2 cells in human asthma, highlighting their many guises and functions in asthma, both pathogenic and regulatory, and how these are influenced by the tissue location and disease stage and severity. It also explores how biologics targeting type 2 immune pathways are impacting asthma, and how these have the potential to reveal hitherto underappreciated roles for Th2 cell in lung inflammation.

Enhanced immune responses following heterologous vaccination with self-amplifying RNA and mRNA COVID-19 vaccines.

The optimal vaccination strategy to boost responses in the context of pre-existing immune memory to the SARS-CoV-2 spike (S) glycoprotein is an important question for global public health. To address this, we explored the SARS-CoV-2-specific humoral and cellular immune responses to a novel self-amplifying RNA (saRNA) vaccine followed by a UK authorised mRNA vaccine (BNT162b2) in individuals with and without previous COVID-19, and compared these responses with those who received an authorised vaccine alone. 35 subjects receiving saRNA (saRNA group) as part of the COVAC1 clinical trial and an additional 40 participants receiving an authorised SARS-CoV-2 vaccine only (non-saRNA group) were recruited. Antibody responses were measured by ELISA and a pseudoneutralisation assay for wildtype, Delta and Omicron variants. Cellular responses were measured by IFN-Æ´ ELISpot and an activation induced marker (AIM) assay. Approximately 50% in each group had previous COVID-19 prior to vaccination, confirmed by PCR or antibody positivity on ELISA. All of those who received saRNA subsequently received a full course of an authorised vaccine. The majority (83%) of those receiving saRNA who were COVID-19 naïve at baseline seroconverted following the second dose, and those with previous COVID-19 had an increase in antibody titres two weeks following saRNA vaccination (median 27-fold), however titres were lower when compared to mRNA vaccination. Two weeks following the 2nd authorised mRNA vaccine dose, binding and neutralising antibody titres were significantly higher in the saRNA participants with previous COVID-19, compared to non-saRNA, or COVID-19 naive saRNA participants. Cellular responses were again highest in this group, with a higher proportion of spike specific CD8+ than CD4+ T cells when compared to those receiving the mRNA vaccine only. These findings suggest an immunological benefit of increased antigen exposure, both from natural infection and vaccination, particularly evident in those receiving heterologous vaccination with saRNA and mRNA.

Hematopoietic Prostaglandin D2 Synthase Controls Tfh/Th2 Communication and Limits Tfh Antitumor Effects.

T follicular helper (Tfh) cells are a subset of CD4+ T cells essential in immunity and have a role in helping B cells produce antibodies against pathogens. However, their role during cancer progression remains unknown. The mechanism of action of Tfh cells remains elusive because contradictory data have been reported on their protumor or antitumor responses in human and murine tumors. Like Tfh cells, Th2 cells are also involved in humoral immunity and are regularly associated with tumor progression and poor prognosis, mainly through their secretion of IL4. Here, we showed that Tfh cells expressed hematopoietic prostaglandin D2 (PGD2) synthase in a pSTAT1/pSTAT3-dependent manner. Tfh cells produced PGD2, which led to recruitment of Th2 cells via the PGD2 receptor chemoattractant receptor homologous molecule expressed on Th type 2 cells (CRTH2) and increased their effector functions. This cross-talk between Tfh and Th2 cells promoted IL4-dependent tumor growth. Correlation between Th2 cells, Tfh cells, and hematopoietic PGD2 synthase was observed in different human cancers and associated with outcome. This study provides evidence that Tfh/Th2 cross-talk through PGD2 limits the antitumor effects of Tfh cells and, therefore, could serve as a therapeutic target.

Immuno-proteomic profiling reveals aberrant immune cell regulation in the airways of individuals with ongoing post-COVID-19 respiratory disease.

Some patients hospitalized with acute COVID-19 suffer respiratory symptoms that persist for many months. We delineated the immune-proteomic landscape in the airways and peripheral blood of healthy controls and post-COVID-19 patients 3 to 6 months after hospital discharge. Post-COVID-19 patients showed abnormal airway (but not plasma) proteomes, with an elevated concentration of proteins associated with apoptosis, tissue repair, and epithelial injury versus healthy individuals. Increased numbers of cytotoxic lymphocytes were observed in individuals with greater airway dysfunction, while increased B cell numbers and altered monocyte subsets were associated with more widespread lung abnormalities. A one-year follow-up of some post-COVID-19 patients indicated that these abnormalities resolved over time. In summary, COVID-19 causes a prolonged change to the airway immune landscape in those with persistent lung disease, with evidence of cell death and tissue repair linked to the ongoing activation of cytotoxic T cells.

Autoantibodies are present in the bronchoalveolar lavage but not circulation in patients with fibrotic interstitial lung disease.

BACKGROUND: Fibrotic interstitial lung disease (fILD) has previously been associated with the presence of autoantibody. While studies have focused on systemic autoimmunity, the role of local autoantibodies in the airways remains unknown. We therefore extensively characterised the airway and peripheral autoantibody profiles in patients with fILD, and assessed association with disease severity and outcome. METHODS: Bronchoalveolar lavage (BAL) fluid was collected from a cohort of fILD patients and total BAL antibody concentrations were quantified. An autoantigen microarray was used to measure IgG and IgA autoantibodies against 122 autoantigens in BAL from 40 idiopathic pulmonary fibrosis (IPF), 20 chronic hypersensitivity pneumonitis (CHP), 20 connective tissue disease-associated ILD (CTD-ILD) patients and 20 controls. RESULTS: A subset of patients with fILD but not healthy controls had a local autoimmune signature in their BAL that was not present systemically, regardless of disease. The proportion of patients with IPF with a local autoantibody signature was comparable to that of CTD-ILD, which has a known autoimmune pathology, identifying a potentially novel subset of patients. The presence of an airway autoimmune signature was not associated with reduced survival probability or changes in lung function in the cohort as a whole. Patients with IPF had increased BAL total IgA and IgG1 while subjects with CHP had increased BAL IgA, IgG1 and IgG4. In patients with CHP, increased BAL total IgA was associated with reduced survival probability. CONCLUSION: Airway autoantibodies that are not present systemically identify a group of patients with fILD and the mechanisms by which these autoantibodies contribute to disease requires further investigation.

T Follicular Helper Cells in Asthma Through Murine Models of Allergic Airway Disease.

The development of allergen-specific IgE is one of the hallmark symptoms of allergic diseases, including asthma. T follicular helper cells (TFH) are a subset of CD4+ T cells that play a critical role in T-dependent antibody responses, including the generation of allergen-specific IgE. However, the role that TFH play in the pathogenesis of allergic disease is not completely understood especially as TFH produce IL-4 and IL-21 which are known to promote and prevent class switch recombination to IgE respectively. Here we describe methods of investigating TFH biology in the context of allergic airway inflammation, including how to set up mouse models of allergic airway disease, flow cytometric analysis of mouse TFH and detection of allergic-specific antibodies.

Enhanced IL-2 in early life limits the development of TFH and protective antiviral immunity.

T follicular helper cell (TFH)-dependent antibody responses are critical for long-term immunity. Antibody responses are diminished in early life, limiting long-term protective immunity and allowing prolonged or recurrent infection, which may be important for viral lung infections that are highly prevalent in infancy. In a murine model using respiratory syncytial virus (RSV), we show that TFH and the high-affinity antibody production they promote are vital for preventing disease on RSV reinfection. Following a secondary RSV infection, TFH-deficient mice had significantly exacerbated disease characterized by delayed viral clearance, increased weight loss, and immunopathology. TFH generation in early life was compromised by heightened IL-2 and STAT5 signaling in differentiating naive T cells. Neutralization of IL-2 during early-life RSV infection resulted in a TFH-dependent increase in antibody-mediated immunity and was sufficient to limit disease severity upon reinfection. These data demonstrate the importance of TFH in protection against recurrent RSV infection and highlight a mechanism by which this is suppressed in early life.

Follicular helper-T cells restore CD8+-dependent antitumor immunity and anti-PD-L1/PD-1 efficacy.

BACKGROUND: T follicular helper cells (Tfh) are essential to shape B cell response during germinal center formation. Tfh accumulation has been reported in various human cancers, with positive or negative prognostic roles. However, the mechanisms explaining the accumulation of Tfh and their role in cancer remain obscure. METHODS: In vitro differentiated and mouse cell sorted Tfh phenotype was evaluated by flow cytometry and quantitative PCR (qPCR). Antitumor effect of Tfh was evaluated by adoptive transfer in different tumor-bearing mice models. The involvement of immune cells, cytokines and chemokines was evaluated, using depleting antibodies. Chemokines and cytokines expression and production were evaluated by qPCR and ELISA. In human, the impact of immune cells and chemokines on survival was evaluated by analyzing transcriptomic data from public databases and from our own patient cohorts. RESULTS: In this study, we show that Tfh exert an antitumor immune effect in a CD8+-dependent manner. Tfh produce interleukin-21, which sustains proliferation, viability, cytokine production and cytotoxic functions of exhausted T cells. The presence of Tfh is required for efficacy of antiprogrammed cell death ligand-1 therapy. Tfh accumulate in the tumor bed and draining lymph nodes in different mouse cancer models. This recruitment is due to the capacity of transforming growth factor ß to drive Chemokine (C-X-C motif) Ligand 13 expression, a chemoattractant of Tfh, by intratumor CD8+ T cells. Accumulation of Tfh and exhausted CD8+ T cells predicts cancer outcome in various cancer types. In patients treated with anti-programmed cell death-1 mAb, accumulation of Tfh and CD8+ at the tumor site is associated with outcome. CONCLUSION: This study provides evidence that CD8+/Tfh crosstalk is important in shaping antitumor immune response generated by immunotherapy.

Lung Marginated and Splenic Murine Resident Neutrophils Constitute Pioneers in Tissue-Defense During Systemic E. coli Challenge.

The rapid response of neutrophils throughout the body to a systemic challenge is a critical first step in resolution of bacterial infection such as Escherichia coli (E. coli). Here we delineated the dynamics of this response, revealing novel insights into the molecular mechanisms using lung and spleen intravital microscopy and 3D ex vivo culture of living precision cut splenic slices in combination with fluorescent labelling of endogenous leukocytes. Within seconds after challenge, intravascular marginated neutrophils and lung endothelial cells (ECs) work cooperatively to capture pathogens. Neutrophils retained on lung ECs slow their velocity and aggregate in clusters that enlarge as circulating neutrophils carrying E. coli stop within the microvasculature. The absolute number of splenic neutrophils does not change following challenge; however, neutrophils increase their velocity, migrate to the marginal zone (MZ) and form clusters. Irrespective of their location all neutrophils capturing heat-inactivated E. coli take on an activated phenotype showing increasing surface CD11b. At a molecular level we show that neutralization of ICAM-1 results in splenic neutrophil redistribution to the MZ under homeostasis. Following challenge, splenic levels of CXCL12 and ICAM-1 are reduced allowing neutrophils to migrate to the MZ in a CD29-integrin dependent manner, where the enlargement of splenic neutrophil clusters is CXCR2-CXCL2 dependent. We show directly molecular mechanisms that allow tissue resident neutrophils to provide the first lines of antimicrobial defense by capturing circulating E. coli and forming clusters both in the microvessels of the lung and in the parenchyma of the spleen.

Overlapping and distinct features of viral and allergen immunity in the human lung.

Immunity in the human respiratory tract is provided by a diverse range of tissue-resident cells, including specialized epithelial and macrophage populations and a network of innate and innate-like lymphocytes, such as natural killer cells, innate lymphoid cells, and invariant T cells. Lung-resident memory T and B cells contribute to this network following initial exposure to antigenic stimuli. This review explores how advances in the study of human immunology have shaped our understanding of this resident immune network and its response to two of the most commonly encountered inflammatory stimuli in the airways: viruses and allergens. It discusses the many ways in which pathogenic infection and allergic inflammation mirror each other, highlighting the key checkpoints at which they diverge and how this can result in a lifetime of allergic exacerbation versus protective anti-viral immunity.

Immunological fortification at our barrier organs: Protecting us as we age.

Our barrier surfaces are fundamental in protecting us from the outside world and segregating key biological processes. The immunological fortifications found at these sites therefore possess many distinct qualities, which are discussed in Immunology's series of reviews on Barrier Immunity. Together these reviews showcase novel biological processes identified through the use of state-of-the-art technologies, and specifically highlight how these change throughout our lives.

Monocarboxylate transporter 1 blockade with AZD3965 inhibits lipid biosynthesis and increases tumour immune cell infiltration.

BACKGROUND: Monocarboxylate transporter 1 (MCT1) is a regulator of cell metabolism and a therapeutic target for cancer treatment. Understanding the changes in tumour function accompanying MCT1 inhibition will better characterise the anti-tumour effects of MCT1 inhibitors, potentially enabling the identification of pharmacodynamic biomarkers for the clinical development of these agents. METHODS: We assessed the impact of the MCT1 inhibitor AZD3965 on tumour metabolism and immune cell infiltration as key determinants of tumour biological function in the MCT1-dependent Raji B cell lymphoma model. RESULTS: Treatment of Raji xenograft-bearing severe combined immunodeficiency mice with AZD3965 led to inhibition of tumour growth paralleled with a decrease in tumour choline, as detected by non-invasive in vivo proton nuclear magnetic resonance spectroscopy. This effect was attributed to inhibition of phosphocholine de novo synthesis following decreased choline kinase α protein and messenger RNA expression that correlated with the AZD3965-induced build-up in intracellular lactate. These changes were concomitant with increased tumour immune cell infiltration involving dendritic and natural killer cells. CONCLUSIONS: Our data provide new insights into the metabolic and cellular changes that occur in the tumour microenvironment following MCT1 blockade, which may contribute to the anti-tumour activity of AZD3965 and could have potential as pharmacodynamic biomarkers of MCT1 inhibition.

T Cell-Intrinsic IL-6R Signaling Is Required for Optimal ICOS Expression and Viral Control during Chronic Infection.

The pleiotropic cytokine IL-6 plays an integral role not only in innate inflammatory responses but also in the activation and differentiation of lymphocyte subsets. In this study, by using a conditional knockout (cKO) model with selective IL-6 receptor deletion in T cells (IL-6R-cKO), we demonstrated that T cell-specific IL-6R signaling is essential for viral control during persistent lymphocytic choriomeningitis virus clone 13 infection. Strikingly, we observed that in contrast to previous studies with ubiquitous IL-6 deletion or blockade, specific IL-6R deletion in T cells did not affect T follicular helper (Tfh) cell accumulation unless IL-6R-deficient T cells were competing with wild-type cells in mixed bone marrow chimeras. In contrast, Tfh cells from IL-6R-cKO-infected mice exhibited reduced ICOS expression in both chimeric and nonchimeric settings, and this sole identifiable Tfh defect was associated with reduced germinal centers, compromised Ig switch and low avidity of lymphocytic choriomeningitis virus-specific Abs despite intact IL-6R expression in B cells. We posit that IL-6R cis-signaling is absolutely required for appropriate ICOS expression in Tfh cells and provides a competitive advantage for Tfh accumulation, enabling generation of optimal B cell and Ab responses, and ultimately viral control during in vivo chronic infection.