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Background: Dysregulation of the complement system is involved in development of age-related macular degeneration (AMD). The complement cascade is regulated by membrane bound complement regulatory proteins (Cregs) on mononuclear leukocytes among others. This study aims to investigate systemic complement proteins and Cregs in AMD stages and their association with treatment response in neovascular AMD (nAMD)., Methods: In this clinical prospective study, treatment-naïve patients with nAMD, intermediate AMD (iAMD) and healthy controls were recruited and systemic complement proteins C3, C3a and C5a were investigated with electrochemiluminescence immunoassays, and Creg expression (CD35, CD46 and CD59) on T cells (CD4 + and CD8+) and monocytes (classical, intermediate and non-classical) investigated with flow cytometry. Treatment response in nAMD patients was evaluated after loading dose and after one year, and categorized as good, partial or poor. Complement proteins and Creg expression levels were compared between healthy controls, iAMD and nAMD, as well as between good, partial and poor nAMD treatment response groups. Polymorphisms in the CFH and ARMS2 genes were analyzed and compared to complement proteins and Creg expression levels in nAMD patients., Results: One hundred patients with nAMD, 34 patients with iAMD and 61 healthy controls were included. 94 nAMD patients completed the 1-year follow-up. Distribution of treatment response in nAMD was 61 (65%) good, 26 (28%) partial, and 7 (7%) poor responders. The distribution of 1-year treatment response was 50 (53%) good, 33 (36%) partial, and 11 (11%) poor responders. The concentrations of systemic C3, C3a, and the C3a/C3-ratio were significantly increased in patients with nAMD compared to healthy controls (P < 0.001, P = 0.002, and P = 0.035, respectively). Systemic C3 was also increased in iAMD compared to healthy controls (P = 0.031). The proportion of CD46 + CD4 + T cells and CD59 + intermediate monocytes were significantly decreased in patients with nAMD compared to healthy controls (P = 0.018 and P = 0.042, respectively). The post-loading dose partial treatment response group had significantly lower concentrations of C3a and C5a compared to the good response group (P = 0.005 and P = 0.042, respectively). The proportion of CD35 + monocytes was significantly lower in the 1-year partial response group compared to the 1-year good response group (P = 0.039). High-risk CFH genotypes in nAMD patients was associated with increased C3a, C3a/C3-ratio, and expression levels of CD35 + CD8 + T cells and CD46 + classical monocytes, while expression level of CD46 + non-classical monocytes was decreased., Conclusion: Elevated concentrations of systemic complement proteins were found in patients with iAMD and nAMD. Decreased Creg expression levels were found in patients with nAMD. Partially responding nAMD patients had a dysregulated complement system and Cregs compared to good responders., (© 2024. The Author(s).)

Aging changes the responsiveness of our immune defense, and this decline in immune reactivity plays an important role in the increased susceptibility to infections that marks progressing age. Aging is also the most pronounced risk factor for development of age-related macular degeneration (AMD), a disease that is characterized by dysfunctional retinal pigment epithelial (RPE) cells and loss of central vision. We have previously shown that acute systemic viral infection has a large impact on the retina in young mice, leading to upregulation of chemokines in the RPE/choroid (RPE/c) and influx of CD8 T cells in the neuroretina. In this study, we sought to investigate the impact of systemic infection on the RPE/c in aged mice to evaluate whether infection in old age could play a role in the pathogenesis of AMD. We found that systemic infection in mice led to upregulation of genes from the crystallin family in the RPE/c from aged mice, but not in the RPE/c from young mice. Crystallin alpha A (CRYAA) was the most upregulated gene, and increased amounts of CRYAA protein were also detected in the aged RPE/c. Increased CRYAA gene and protein expression has previously been found in drusen and choroid from AMD patients, and this protein has also been linked to neovascularization. Since both drusen and neovascularization are important hallmarks of advanced AMD, it is interesting to speculate if upregulation of crystallins in response to infection in old age could be relevant for the pathogenesis of AMD., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Blindness or vision loss due to neuroretinal and photoreceptor degeneration affects millions of individuals worldwide. In numerous neurodegenerative diseases, including age-related macular degeneration, dysregulated immune response-mediated retinal degeneration has been found to play a critical role in the disease pathogenesis. To better understand the pathogenic mechanisms underlying the retinal degeneration, we used a mouse model of systemic immune activation where we infected mice with lymphocytic choriomeningitis virus (LCMV) clone 13. Here, we evaluated the effects of LCMV infection and present a comprehensive discovery-based proteomic investigation using tandem mass tag (TMT) labeling and high-resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS). Changes in protein regulation in the posterior part of the eye, neuroretina, and RPE/choroid were compared to those in the spleen as a secondary lymphoid organ and to the kidney as a non-lymphoid but encapsulated organ at 1, 8, and 28 weeks of infection. Using bioinformatic tools, we found several proteins responsible for maintaining normal tissue homeostasis to be differentially regulated in the neuroretina and the RPE/choroid during the degenerative process. Additionally, in the organs we observed, several important protein pathways contributing to cellular homeostasis and tissue development were perturbed and associated with LCMV-mediated inflammation, promoting disease progression. Our findings suggest that the response to a systemic chronic infection differs between the neuroretina and the RPE/choroid, and the processes induced by chronic systemic infection in the RPE/choroid are not unlike those induced in non-immune-privileged organs such as the kidney and spleen. Overall, our data provide detailed insight into several molecular mechanisms of neuroretinal degeneration and highlight various novel protein pathways that further suggest that the posterior part of the eye is not an isolated immunological entity despite the existence of neuroretinal immune privilege., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Khan, Steffensen, Paskeviciute, Abduljabar, Sørensen, Vorum, Nissen and Honoré.)

Purpose: Geographic atrophy (GA) secondary to age-related macular degeneration is a progressive retinal degenerative disease. Systemic chemokine receptors and known risk-associated single-nucleotide polymorphisms have been associated with GA pathogenesis. Because halting progression is pivotal for patients, we investigated the association of candidate chemokine receptors and progression rate (PR) of atrophic lesions in patients with GA., Methods: This prospective observational study conducted at a single center included 85 patients with GA and 45 healthy controls. Patients were followed up after 13 months on average. Serial fundus autofluorescence images were used to determine the PR of atrophic lesions. The proportion of chemokine receptors on peripheral lymphocytes were determined by flow cytometric analysis., Results: Patients with GA had a lower proportion of CCR6 on CD8+T cells compared to healthy controls. Importantly, the proportion of CCR6 on CD4+T cells was lower in patients with fast GA progression compared to patients with slow progression of disease, suggesting that dysregulation of CCR6 could be involved in progression of GA. We also found that GA patients had a markedly higher percentage of CCR5 on CD4+ and CD8+T cells compared to healthy controls. After stratification according to ARMS2 polymorphism, we found a significantly lower level of CCR5 on CD8+T cells among patients with high-risk genotypes compared with patients with the low-risk genotype., Conclusions: Our study finds that chemokine receptors are dysregulated in patients with GA and that CCR6 might be involved in GA progression, making it a potential target for intervention.

During recent years, evidence has emerged that immune privileged sites such as the CNS and the retina may be more integrated in the systemic response to infection than was previously believed. In line with this, it was recently shown that a systemic acute virus infection leads to infiltration of CD8 T cells in the brains of immunocompetent mice. In this study, we extend these findings to the neurological tissue of the eye, namely the retina. We show that an acute systemic virus infection in mice leads to a transient CD8 T cell infiltration in the retina that is not directed by virus infection inside the retina. CD8 T cells were found throughout the retinal tissue, and had a high expression of CXCR6 and CXCR3, as also reported for tissue residing CD8 T cells in the lung and liver. We also show that the pigment epithelium lining the retina expresses CXCL16 (the ligand for CXCR6) similar to epithelial cells of the lung. Thus, our results suggest that the retina undergoes immune surveillance during a systemic infection, and that this surveillance appears to be directed by mechanisms similar to those described for non-privileged tissues., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Paskeviciute, Chen, Xu, Honoré, Vorum, Sørensen, Christensen, Thomsen, Nissen and Steffensen.)

Background: This randomized, double-blind trial was conducted to determine the optimal dose for clinical efficacy of the SQ tree SLIT-tablet. An environmental exposure chamber (EEC) was used to reduce variability of allergen exposure and allow investigation of symptom reduction towards different species from the birch homologous group in separate EEC sessions., Methods: Eligible subjects (N = 219) were randomized to receive treatment with placebo or the SQ tree SLIT-tablet (2, 7, or 12 DU) for 24 weeks. EEC pollen challenges were conducted outside the birch pollen season and included four birch and two oak EEC sessions. The primary efficacy endpoint was the average allergic rhinoconjunctivitis (ARC) total symptom score (TSS) after 24 weeks of treatment., Results: There was a statistically significantly lower TSS during the 24-week birch EEC session for 7 DU and 12 DU compared to placebo with relative differences of 24% (P = 0.03) and 25% (P = 0.02). For the 24-week oak EEC session, there was a statistically significant difference for 12 DU (24%, P = 0.03). IgE and IgG4 measurements supported these findings and demonstrated cross-reactivity to all other species within the birch homologous group. Treatment was well-tolerated with the most frequently reported adverse reactions being the local reactions in the oral cavity of mild-to-moderate severity., Conclusion: This trial demonstrates that the SQ tree SLIT-tablet reduce ARC symptoms triggered by birch or oak pollen. The optimal dose for further development was 12 DU. Clinical and immunological findings suggest that the tablet may be used to treat allergies to all species within the birch homologous group., (© 2018 The Authors. Allergy Published by John Wiley & Sons Ltd.)

Defining correlates of T cell mediated protection is important in order to accelerate the development of efficient T cell based vaccines conferring long-term immunity. Extensive studies have provided important insight regarding the characteristics and functional properties of the effector and memory CD8 T cells induced by viral vector based vaccines. However, long-term protection has been difficult to achieve with T cell inducing vaccines, and the determinants underlying this loss in protection over time are still not fully defined. In this study we analyzed different parameters of the CD8 T cell response as a function of time after vaccination with a human serotype 5 adenovector expressing the glycoprotein (GP) of LCMV tethered to the MHC class II-associated invariant chain. Using this vector we have previously found that CD8 T cells mediate protection from challenge with GP-expressing Listeria monocytogenes at 60 days post vaccination, but only little protection after further 60 days, and we now confirm this observation. A comparison of vaccine-primed CD8 T cells early and late after vaccination revealed a minor decline in the overall numbers of antigen specific memory CD8 T cells during this interval. More importantly, we also observed phenotypic changes over time with a distinct decline in the frequency and number of KLRG1 + CD8 T cells, and, notably, adoptive transfer studies confirmed that memory CD8 T cells expressing KLRG1 are central to protection from systemic L. monocytogenes infection. Together these findings imply that multiple factors including changes in memory T cell numbers and phenotypic composition over time influence the longevity of CD8 T-cell mediated protection., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Purpose: The SQ tree sublingual immunotherapy (SLIT)-tablet containing allergen extracts with the major allergen Bet v 1 from birch pollen is currently being developed for the treatment of tree pollen-induced allergic rhinitis/conjunctivitis with or without asthma. The aim of this Phase II trial was to investigate the dose-related efficacy and safety of the SQ tree SLIT-tablet., Methods: This study was a randomized, parallel-group, double-blind, placebo-controlled, multi-national trial conducted in Europe. A total of 637 participants were randomized equally to receive placebo or treatment with the SQ tree SLIT-tablet in doses of 0.5, 1, 2, 4, 7, or 12 development units (DU). Treatment was initiated ~16 weeks before onset of the 2013 birch pollen season (BPS) and was continued throughout the BPS with a total duration of at least 6 months. During the BPS and tree pollen season (TPS), subjects assessed rhinoconjunctivitis symptoms and medication use on a daily basis in an electronic diary; weekly assessments of rhinoconjunctivitis quality of life were also made., Findings: Analysis of the average daily symptom score during the BPS and the TPS showed that the difference between active treatment and placebo was statistically significant for the 7 DU group (BPS, P = 0.02; TPS, P = 0.03), with no clear dose-response relationship. All doses of the SQ tree SLIT-tablet induced changes from baseline in birch-specific IgE and IgG 4 that were statistically significant compared with placebo at all time points assessed (P < 0.0001) with a clear dose-response relationship for birch specific IgG 4 . In general, the SQ tree SLIT-tablet was well tolerated, with the majority of treatment-related adverse events (≥95%) being mild or moderate in severity. The most frequently reported treatment-related adverse events were generally related to the sublingual administration of the tablet (ie, they occurred in the oral cavity)., Implications: The results from this trial suggest that the SQ tree SLIT-tablet in doses up to 12 DU has a tolerability profile suitable for at-home administration. The immunomodulatory changes indicate a dose-response relationship, but clinical efficacy parameters were inconclusive, probably due to low pollen counts, emphasizing the importance of pollen exposure for the outcome of a pollen allergy immunotherapy trial. EudraCT no: 2012-000031-59., (Copyright © 2018 Elsevier HS Journals, Inc. All rights reserved.)

Purpose: The tree pollen sublingual immunotherapy (SLIT)-tablet (ALK, Denmark) is being developed for the treatment of tree pollen induced allergic rhinitis with or without conjunctivitis. The objective of this Phase I trial was to investigate the tolerability and acceptable dose range of the SQ tree SLIT-tablet in adults with allergic rhinoconjunctivitis., Methods: The trial was a randomized, double-blind, placebo-controlled, dose escalation Phase I trial that included 70 adults (aged 19-61 years) with birch pollen-induced rhinoconjunctivitis with or without mild to moderate asthma. The trial included 6 different dosage groups that were randomized 3:1 to active treatment or placebo once daily for 28 days. Adverse events (AEs) were coded in the Medical Dictionary for Regulatory Activities by medically qualified personnel. Immunologic assessments included IgE and IgE-blocking factor., Findings: Most (96%) reported AEs were mild, and only 5 severe events (0.2%) were reported. The most frequently reported investigational medicinal product-related AEs were oral pruritus, ear pruritus, mouth edema, sensation of foreign body, throat irritation, pharyngolaryngeal pain, dry throat, tongue blistering, eye pruritus, and headache. The trial included doses ranging from 1 to 24 development units (DU), and the mean number of investigational medicinal product-related AEs per participant was highest in the 24 DU group. The 12 and 24 DU doses induced statistically significant changes from baseline compared with placebo in birch specific IgE and IgE-blocking factor., Implications: The trial found that doses up to 12 DU of the SQ tree SLIT tablet have a tolerability profile suitable for at-home administration. An immunomodulatory effect was found for all doses included in the trial, and doses up to 12 DU were thus chosen for further clinical development of the SQ tree SLIT tablet. EudraCT identifier: 2007-003234-42., (Copyright © 2017 Elsevier HS Journals, Inc. All rights reserved.)

Recently, we showed that combined intranasal and subcutaneous immunization with a non-replicating adenoviral vector expressing NP of influenza A, strain PR8, induced long-standing protection against a range of influenza A viruses. However, H-2 b mice challenged with an influenza A strain mutated in the dominant NP 366 epitope were not efficiently protected. To address this problem, we envision the use of a cocktail of adenovectors targeting different internal proteins of influenza A virus. Consequently, we investigated the possibility of using PB1 as a target for an adenovector-based vaccine against influenza A. Our results showed that PB1 is not as immunogenic as the NP protein. However, by tethering PB1 to the murine invariant chain we were able to circumvent this problem and raise quite high numbers of PB1-specific CD8 + T cells in the circulation. Nevertheless, mice immunized against PB1 were not as efficiently protected against influenza A challenge as similarly NP-vaccinated animals. The reason for this is not a difference in the quality of the primed cells, nor in functional avidity. However, under similar conditions of immunization fewer PB1-specific cells were recruited to the airways, and surface expression of the dominant PB1 peptide, PB1 703 , was less stable than in the case of NP 366 ., Competing Interests: Together with the University of Copenhagen and Peter J Holst, the authors JPC and ART hold a patent regarding the invariant chain fusion technology.

Background: Citrullination catalysed by peptidylarginine deiminases (PADs) plays an important pathogenic role in anti-citrullinated protein antibody (ACPA)-positive rheumatoid arthritis (RA) and, possibly, several other inflammatory diseases. Non-physiological reducing agents such as dithiothreitol (DTT) are normally added to the reaction buffer when determining PAD activity in vitro. We investigated the ability of reduced glutathione (GSH), the most abundant intracellular small-molecule thiol in vivo, to activate PADs., Methods: Activity of recombinant human (rh) PAD2 and PAD4, PADs contained in synovial fluid (SF) samples from RA patients and PADs released from phorbol 12-myristate 13-acetate (PMA)-stimulated cells was measured using an in-house PAD activity assay detecting citrullination of fibrinogen., Results: No activity of rhPAD2, rhPAD4 or PADs within SF was observed without addition of an exogenous reducing agent. Activity of both recombinant and SF PAD was observed in the presence of 1 mM DTT or 10-15 mM GSH. Following stimulation with PMA, human isolated leucocytes, but not mononuclear cells, released enzymatically active PAD, the activity of which was abolished upon pre-incubation of the cells with the glutathione reductase inhibitor 2-AAPA. No PAD activity was observed in the corresponding supernatants, but addition of exogenous GSH restored activity., Conclusions: Catalytic activity of PAD requires reducing conditions. GSH meets this requirement at concentrations comparable with those found within cells. Active PAD, reduced by GSH, is released from PMA-stimulated granulocytes, but becomes inactivated in the extracellular space.

As a result of the difficulties in making efficient vaccines against genetically unstable viruses such as HIV, it has been suggested that future vaccines should preferentially target subdominant epitopes, the idea being that this should allow a greater breadth of the induced T cell response and, hence, a greater efficiency in controlling escape variants. However, to our knowledge the evidence supporting this concept is limited at best. To improve upon this, we used the murine lymphocytic choriomeningitis virus model and adenoviral vectors to compare a vaccine expressing unmodified Ag to a vaccine expressing the same Ag without its immunodominant epitope. We found that removal of the dominant epitope allowed the induction of CD8(+) T cell responses targeting at least two otherwise subdominant epitopes. Importantly, the overall magnitude of the induced T cell responses was similar, allowing us to directly compare the efficiency of these vaccines. Doing this, we observed that mice vaccinated with the vaccine expressing unmodified Ag more efficiently controlled an acute viral challenge. In the course of a more chronic viral infection, mice vaccinated using the vaccine targeting subdominant epitopes caught up with the conventionally vaccinated mice, and analysis of the breadth of the CD8(+) T cell response revealed that this was notably greater in the former mice. However, under the conditions of our studies, we never saw any functional advantage of this. This may represent a limitation of our model, but clearly our findings underscore the importance of carefully weighing the pros and cons of changes in epitope targeting before any implementation., (Copyright © 2016 by The American Association of Immunologists, Inc.)

The threat from unpredictable influenza virus pandemics necessitates the development of a new type of influenza vaccine. Since the internal proteins are highly conserved, induction of T cells targeting these antigens may provide the solution. Indeed, adenoviral (Ad) vectors expressing flu nucleoprotein have previously been found to induce short-term protection in mice. In this study we confirm that systemic (subcutaneous (s.c.) immunization rapidly induced heterosubtypic protection predominantly mediated by CD8 T cells, but within three months clinical protection completely disappeared. Local (intranasal (i.n.)) immunization elicited delayed, but more lasting protection despite relatively inefficient immunization. However, by far, the most robust protection was induced by simultaneous, combined (i.n. + s.c.) vaccination, and, notably, in this case clinical protection lasted at least 8 months without showing any evidence of fading. Interestingly, the superior ability of the latter group to resist reinfection correlated with a higher number of antigen-specific CD8 T cells in the spleen. Thus, detailed analysis of the underlying CD8 T cell responses highlights the importance of T cells already positioned in the lungs prior to challenge, but at the same time underscores an important back-up role for circulating antigen-specific cells with the capacity to expand and infiltrate the infected lungs.

The attenuated yellow fever (YF) vaccine (YF-17D) was developed in the 1930s, yet little is known about the protective mechanisms underlying its efficiency. In this study, we analyzed the relative contribution of cell-mediated and humoral immunity to the vaccine-induced protection in a murine model of YF-17D infection. Using different strains of knockout mice, we found that CD4(+) T cells, B cells, and Abs are required for full clinical protection of vaccinated mice, whereas CD8(+) T cells are dispensable for long-term survival after intracerebral challenge. However, by analyzing the immune response inside the infected CNS, we observed an accelerated T cell influx into the brain after intracerebral challenge of vaccinated mice, and this T cell recruitment correlated with improved virus control in the brain. Using mice deficient in B cells we found that, in the absence of Abs, YF vaccination can still induce some antiviral protection, and in vivo depletion of CD8(+) T cells from these animals revealed a pivotal role for CD8(+) T cells in controlling virus replication in the absence of a humoral response. Finally, we demonstrated that effector CD8(+) T cells also contribute to viral control in the presence of circulating YF-specific Abs. To our knowledge, this is the first time that YF-specific CD8(+) T cells have been demonstrated to possess antiviral activity in vivo., (Copyright © 2015 by The American Association of Immunologists, Inc.)

We have previously shown that for the majority of antigens, adenoviral vaccines expressing the target antigen fused to the MHC associated invariant chain (Ii) induce an accelerated, augmented, and prolonged transgene-specific CD8(+) T-cell response. Here we describe a new adenoviral vaccine vector approach where the target antigen fused to Ii is expressed from the adenoviral E1 region and IL-2 is expressed from the E3 region. Immunization of mice with this new vector construct resulted in an augmented primary effector CD8(+) T-cell response. Furthermore, in a melanoma model we observed significantly prolonged tumor control in vaccinated wild type (WT) mice. The improved tumor control required antigen-specific cells, since no tumor control was observed, unless the melanoma cells expressed the vaccine targeted antigen. We also tested our new vaccine in immunodeficient (CD80/86 deficient) mice. Following vaccination with the IL-2 expressing construct, these mice were able to raise a delayed but substantial CD8(+) T-cell response, and to control melanoma growth nearly as efficaciously as similarly vaccinated WT mice. Taken together, these results demonstrate that current vaccine vectors can be improved and even tailored to meet specific demands: in the context of therapeutic vaccination, the capacity to promote an augmented effector T-cell response.

Unlabelled: Suppressors of cytokine signaling (SOCS) proteins are intracellular proteins that inhibit cytokine signaling in a variety of cell types. A number of viral infections have been associated with SOCS upregulation; however, not much is known about the mechanisms regulating SOCS expression during viral infection. In this study, we used two pathologically distinct intracerebral (i.c.) infection models to characterize temporal and spatial aspects of SOCS expression in the virus-infected central nervous system (CNS), and by employing various knockout mouse models, we sought to identify regulatory mechanisms that may underlie a virus induced upregulation of SOCS in the CNS. We found that i.c. infection with either lymphocytic choriomeningitis virus (LCMV) or yellow fever virus (YF) results in gradual upregulation of SOCS1/3 mRNA expression peaking at day 7 postinfection (p.i.). In the LCMV model, SOCS mRNA was expressed in brain resident cells, including astrocytes and some neurons, and for SOCS1 in particular this upregulation was almost entirely mediated by gamma interferon (IFN-γ) produced by infiltrating T cells. After infection with YF, we also found SOCS expression to be upregulated in brain resident cells with a peak on day 7 p.i., but in this model, the upregulation was only partially dependent on IFN-γ and T cells, indicating that at least one other mediator was involved in the upregulation of SOCS following YF infection. We conclude that virus-induced inflammation of the CNS is associated with upregulation of SOCS1/3 mRNA expression in brain resident cells and that at least two distinctive pathways can lead to this upregulation., Importance: In the present report, we have studied the induction of SOCS1 and SOCS3 expression in the context of virus-induced CNS infection. We found that both a noncytolytic and a cytolytic virus induce marked upregulation of SOCS1 and -3 expression. Notably, the kinetics of the observed upregulation follows that of activity within proinflammatory signaling pathways and, interestingly, type II interferon (IFN), which is also a key inducer of inflammatory mediators, seems to be essential in initiating this counterinflammatory response. Another key observation is that not only cells of the immune system but also CNS resident cells are actively involved in both the pro- and the counterinflammatory immune circuits; thus, for example, astrocytes upregulate both C-X-C-motif chemokine 10 (CXCL10) and SOCS when exposed to type II IFN in vivo., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Adenoviral vectors have long been forerunners in the development of effective CD8 T cell-based vaccines; therefore, it is imperative that we understand the factors controlling the induction of robust and long-lasting transgene-specific immune responses by these vectors. In this study, we investigated the organ sites, molecules, and cell subsets that play a critical role in the priming of transgene-specific CD8 T cells after vaccination with a replication-deficient adenoviral vector. Using a human adenovirus serotype 5 (Ad5) vector and genetically engineered mice, we found that CD8(+) and/or CD103(+) dendritic cells in the draining lymph node played a critical role in the priming of Ad5-induced CD8 T cell responses. Moreover, we found that CD80/86, but not CD28, was essential for efficient generation of both primary effectors and memory CD8 T cells. Interestingly, the lack of CD28 expression resulted in a delayed primary response, whereas memory CD8 T cells generated in CD28-deficient mice appeared almost normal in terms of both phenotype and effector cytokine profile, but they exhibited a significantly reduced proliferative capacity upon secondary challenge while retaining immediate in vivo effector capabilities: in vivo cytotoxicity and short-term in vivo protective capacity. Overall, our data point to an absolute requirement for professional APCs and the expression of the costimulatory molecules CD80/86 for efficient CD8 T cell priming by adenoviral vectors. Additionally, our results suggest the existence of an alternative receptor for CD80/86, which may substitute, in part, for CD28., (Copyright © 2014 by The American Association of Immunologists, Inc.)

The use of replication-deficient adenoviruses as vehicles for transfer of foreign genes offers many advantages in a vaccine setting, eliciting strong cellular immune responses involving both CD8(+) and CD4(+) T cells. Further improving the immunogenicity, tethering of the inserted target Ag to MHC class II-associated invariant chain (Ii) greatly enhances both the presentation of most target Ags, as well as overall protection against viral infection, such as lymphocytic choriomeningitis virus (LCMV). The present study extends this vaccination concept to include protection against intracellular bacteria, using Listeria monocytogenes as a model organism. Protection in C57BL/6 mice against recombinant L. monocytogenes expressing an immunodominant epitope of the LCMV glycoprotein (GP33) was greatly accelerated, augmented, and prolonged following vaccination with an adenoviral vaccine encoding GP linked to Ii compared with vaccination with the unlinked vaccine. Studies using knockout mice demonstrated that CD8(+) T cells were largely responsible for this protection, which is mediated through perforin-dependent lysis of infected cells and IFN-γ production. Taking the concept a step further, vaccination of C57BL/6 (L. monocytogenes-resistant) and BALB/c (L. monocytogenes-susceptible) mice with adenoviral vectors encoding natural L. monocytogenes-derived soluble Ags (listeriolysin O and p60) revealed that tethering of these Ags to Ii markedly improved the vaccine-induced CD8(+) T cell response to two of three epitopes studied. More importantly, Ii linkage accelerated and augmented vaccine-induced protection in both mouse strains and prolonged protection, in particular that induced by the weak Ag, p60, in L. monocytogenes-susceptible BALB/c mice.

It has been reported that adenovirus (Ad)-primed CD8 T cells may display a distinct and partially exhausted phenotype. Given the practical implications of this claim, we decided to analyze in detail the quality of Ad-primed CD8 T cells by directly comparing these cells to CD8 T cells induced through infection with lymphocytic choriomeningitis virus (LCMV). We found that localized immunization with intermediate doses of Ad vector induces a moderate number of functional CD8 T cells which qualitatively match those found in LCMV-infected mice. The numbers of these cells may be efficiently increased by additional adenoviral boosting, and, importantly, the generated secondary memory cells cannot be qualitatively differentiated from those induced by primary infection with replicating virus. Quantitatively, DNA priming prior to Ad vaccination led to even higher numbers of memory cells. In this case, the vaccination led to the generation of a population of memory cells characterized by relatively low CD27 expression and high CD127 and killer cell lectin-like receptor subfamily G member 1 (KLRG1) expression. These memory CD8 T cells were capable of proliferating in response to viral challenge and protecting against infection with live virus. Furthermore, viral challenge was followed by sustained expansion of the memory CD8 T-cell population, and the generated memory cells did not appear to have been driven toward exhaustive differentiation. Based on these findings, we suggest that adenovirus-based prime-boost regimens (including Ad serotype 5 [Ad5] and Ad5-like vectors) represent an effective means to induce a substantially expanded, long-lived population of high-quality transgene-specific memory CD8 T cells.

The scavenger receptor cysteine-rich (SRCR) superfamily is a group of membrane bound and secreted proteins expressed by cells of the immune system. Several members act as pattern recognition receptors that bind to conserved molecular structures of pathogens. We have previously characterized a member of the SRCR superfamily, mSCART1, which primarily is expressed on a large subset of γδ T cells in mice. Here we report the cloning and characterization of human SCART1 (hSCART1) mainly expressed by CD4(+) and CD8(+) T lymphocytes. The hSCART1 gene maps to chromosome 10, region q26.3, a region that displays synteny to the position of mSCART1 in the murine genome. The primary structure of hSCART1 was established by molecular cloning. The longest cDNA sequence of hSCART1 that was found is 2200bp and encodes a protein composed of a signal peptide, 5 SRCR domains, and an in-frame potential cytoplasmic domain. Shorter splice forms have also been isolated. Quantitative real-time PCR analysis on human blood-fractions has shown that hSCART1 is expressed primarily by CD4(+) and CD8(+) T lymphocytes with either αβ or γδ T cell receptors, and real-time PCR on 22 different human tissues showed high expression of hSCART1 in the small intestine and colon. An antibody raised against an N-terminal hSCART1 peptide stains a subset of cells in the small intestine, stomach, and gall bladder, and it also stains placental villi. In conclusion, the characterization of hSCART1 at the mRNA and protein level suggests that the protein plays a role in the immune system, perhaps as a co-receptor on αβ and γδ T cells., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

Adenoviral vectors have shown a great potential for vaccine development due to their inherent ability to induce potent and protective CD8 T-cell responses. However, a critical issue regarding the use of these vectors is the existence of inhibitory immunity against the most commonly used Ad5 vector in a large part of the human population. We have recently developed an improved adenoviral vaccine vector system in which the vector expresses the transgene tethered to the MHC class II associated invariant chain (Ii). To further evaluate the potential of this system, the concept of pre-existing inhibitory immunity to adenoviral vectors was revisited to investigate whether the inhibition previously seen with the Ad5 vector also applied to the optimized vector system. We found this to be the case, and antibodies dominated as the mechanism underlying inhibitory vector immunity. However, presence of CD8 T cells directed against epitopes in the adenoviral vector seemed to correlate with repression of the induced response in re-vaccinated B-cell deficient mice. More importantly, despite a repressed primary effector CD8 T-cell response in Ad5-immune animals subjected to vaccination, memory T cells were generated that provided the foundation for an efficient recall response and protection upon subsequent viral challenge. Furthermore, the transgene specific response could be efficiently boosted by homologous re-immunization. Taken together, these studies indicate that adenoviral vectors can be used to induce efficient CD8 T-cell memory even in individuals with pre-existing vector immunity.

Therapeutic vaccination with replication deficient adenovirus expressing a viral antigen linked to invariant chain was recently found to markedly delay the growth of B16.F10 melanomas expressing the same antigen; however, complete regression of the tumors was never observed. Here we show that the delay in tumor growth can be converted to complete regression and long-term survival in 30-40% of the mice by a booster vaccination plus combinational treatment with agonistic anti-CD40 monoclonal antibodies (mAb) and anti-CTLA-4 mAb. Regarding the mechanism underlying the improved clinical effect, analysis of the tumor-specific response revealed a significantly prolonged tumor-specific CD8 T cell response in spleens of the mice receiving the combinational treatment compared with mice receiving either treatment individually. Matching this, CD8 T cell depletion completely prevented tumor control. These results indicate that even with a strong tumor vaccine candidate, combinatorial treatment may be required to obtain clinically relevant results., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Surfactant protein D (SP-D) is a pattern recognition molecule of the collectin family of C-type lectins. It is found in the airways and at mucosal surfaces. SP-D is part of the innate immune system where it neutralizes and leads to elimination of microorganisms. It regulates the functions of other innate immune cells, such as macrophages and neutrophils. It also modulates the adaptive immune response by interacting with antigen-presenting cells and T cells. Monoclonal anti-mouse-SP-D antibodies were raised from SP-D deficient mice using recombinant SP-D as antigen. Ten monoclonal antibodies were characterized and validated for use in sandwich enzyme-linked immunosorbent assay (ELISA). Based on two of these, we established an ELISA that allows for measurements of mouse SP-D in various body fluids. The final ELISA was optimized and calibrated with a standard of purified recombinant mouse SP-D, which was analyzed by quantitative amino acid analysis. The ELISA was validated with respect to parallelism, recovery, intra- and inter-assay variation. The practical working range was estimated to be 1.9-200 ng/ml. We measured SP-D in lung lavage to an average concentration of 435 ng/ml (3-ml lavage), and in mouse vaginal fluid (1-ml lavage) to an average concentration of 94 ng/ml, but could not detect SP-D in the serum or plasma of healthy mice (<3.8 ng/ml). With this ELISA, it will be possible to study the regulation of SP-D in various mouse models and upon various stimuli.