Influenza virus infection enhances tumour-specific CD8+ T-cell immunity, facilitating tumour control.

Influenza A virus (IAV) can cause severe respiratory infection leading to significant global morbidity and mortality through seasonal epidemics. Likewise, the constantly increasing number of cancer diseases is a growing problem. Nevertheless, the understanding of the mutual interactions of the immune responses between cancer and infection is still very vague. Therefore, it is important to understand the immunological cross talk between cancer and IAV infection. In several preclinical mouse models of cancer, including melanoma and colorectal cancer, we observed that IAV infection in the lung significantly decreased the tumour burden. Concomitantly, tumour-specific CD8+ T-cells are strongly activated upon infection, both in the tumour tissue and in the lung. CD8+ T-cell depletion during infection reverses the reduced tumour growth. Interestingly, IAV infection orchestrated the migration of tumour-specific CD8+ T-cells from the tumour into the infected lung. Blocking the migration of CD8+ T-cells prevented the anti-tumoural effect. Thus, our findings show that viral respiratory infection has significant impact on the anti-tumour CD8+ T-cell response, which will significantly improve our understanding of the immunological cross talk between cancer and infection.

Neuropilin-1 identifies a subset of highly activated CD8+ T cells during parasitic and viral infections.

Neuropilin-1 (Nrp-1) expression on CD8+ T cells has been identified in tumor-infiltrating lymphocytes and in persistent murine gamma-herpes virus infections, where it interferes with the development of long-lived memory T cell responses. In parasitic and acute viral infections, the role of Nrp-1 expression on CD8+ T cells remains unclear. Here, we demonstrate a strong induction of Nrp-1 expression on CD8+ T cells in Plasmodium berghei ANKA (PbA)-infected mice that correlated with neurological deficits of experimental cerebral malaria (ECM). Likewise, the frequency of Nrp-1+CD8+ T cells was significantly elevated and correlated with liver damage in the acute phase of lymphocytic choriomeningitis virus (LCMV) infection. Transcriptomic and flow cytometric analyses revealed a highly activated phenotype of Nrp-1+CD8+ T cells from infected mice. Correspondingly, in vitro experiments showed rapid induction of Nrp-1 expression on CD8+ T cells after stimulation in conjunction with increased expression of activation-associated molecules. Strikingly, T cell-specific Nrp-1 ablation resulted in reduced numbers of activated T cells in the brain of PbA-infected mice as well as in spleen and liver of LCMV-infected mice and alleviated the severity of ECM and LCMV-induced liver pathology. Mechanistically, we identified reduced blood-brain barrier leakage associated with reduced parasite sequestration in the brain of PbA-infected mice with T cell-specific Nrp-1 deficiency. In conclusion, Nrp-1 expression on CD8+ T cells represents a very early activation marker that exacerbates deleterious CD8+ T cell responses during both, parasitic PbA and acute LCMV infections.

Stroke of Consistency: Streamlining Multicenter Protocols for Enhanced Reproducibility of Infarct Volumes in Preclinical Stroke Research.

BACKGROUND: The discrepancy between experimental research and clinical trial outcomes is a persistent challenge in preclinical studies, particularly in stroke research. A possible factor contributing to this issue is the lack of standardization across experimental stroke models, leading to poor reproducibility in multicenter studies. This study addresses this gap by aiming to enhance reproducibility and the efficacy of multicenter studies through the harmonization of protocols and training of involved personnel. METHODS: We established a set of standard operating procedures for various stroke models and the Neuroscore. These standard operating procedures were implemented across multiple research centers, followed by specialized, in-person training for all participants. We measured the variability in infarct volume both before and after the implementation of these standardized protocols and training sessions. RESULTS: The standardization process led to a significant reduction in variability of infarct volume across different stroke models (40%-50% reduction), demonstrating the effectiveness of our harmonized protocols and training. Additionally, the implementation of the Neuroscore system across centers showed low variability and consistent results up to 28 days poststroke, underscoring its utility in chronic phase evaluations. CONCLUSIONS: The harmonization of protocols and surgeon training significantly reduced variability in experimental outcomes across different centers. This improvement can increase the comparability of data between research groups and enhance the statistical power of multicenter studies. Our findings also establish the Neuroscore as a reliable tool for long-term assessment in stroke research, paving the way for more consistent and impactful multicenter preclinical studies.

Delayed plasma kallikrein inhibition fosters post-stroke recovery by reducing thrombo-inflammation.

Activation of the kallikrein-kinin system promotes vascular leakage, inflammation, and neurodegeneration in ischemic stroke. Inhibition of plasma kallikrein (PK) - a key component of the KKS - in the acute phase of ischemic stroke has been reported to reduce thrombosis, inflammation, and damage to the blood-brain barrier. However, the role of PK during the recovery phase after cerebral ischemia is unknown. To this end, we evaluated the effect of subacute PK inhibition starting from day 3 on the recovery process after transient middle artery occlusion (tMCAO). Our study demonstrated a protective effect of PK inhibition by reducing infarct volume and improving functional outcome at day 7 after tMCAO. In addition, we observed reduced thrombus formation in cerebral microvessels, fewer infiltrated immune cells, and an improvement in blood-brain barrier integrity. This protective effect was facilitated by promoting tight junction reintegration, reducing detrimental matrix metalloproteinases, and upregulating regenerative angiogenic markers. Our findings suggest that PK inhibition in the subacute phase might be a promising approach to accelerate the post-stroke recovery process.

Neuropilin-1 Is Expressed on Highly Activated CD4+ Effector T Cells and Dysfunctional CD4+ Conventional T Cells from Naive Mice.

Neuropilin-1 (Nrp-1) is a well described marker molecule for CD4+Foxp3+ thymus-derived regulatory T cells (Tregs). In addition, a small population of CD4+Foxp3- conventional (conv) T cells expresses Nrp-1 in naive mice, and Nrp-1 expression has been described to be upregulated on activated CD4+ T cells. However, the function of Nrp-1 expression on CD4+ non-Tregs still remains elusive. In this study, we demonstrate that Nrp-1 expression was induced upon stimulation of CD4+Foxp3- T cells in vitro and during an ongoing immune response in vivo. This activation-induced Nrp-1+CD4+ T cell subset (iNrp-1+) showed a highly activated phenotype in terms of elevated CD25 and CD44 expression, enhanced production of proinflammatory cytokines, and increased proliferation compared with the Nrp-1-CD4+ counterpart. In contrast, Nrp-1+CD4+Foxp3- conv T cells from naive mice (nNrp-1+) were dysfunctional. nNrp-1+CD4+ conv T cells upregulated activation-associated molecules to a lesser extent, exhibited impaired proliferation and produced fewer proinflammatory cytokines than Nrp-1-CD4+ conv T cells upon stimulation in vitro. Moreover, the expression of PD-1 and CTLA-4 was significantly higher on nNrp-1+CD4+Foxp3- T cells compared with iNrp-1+CD4+Foxp3- T cells and Nrp-1-CD4+Foxp3- T cells after stimulation and under homeostatic conditions. Strikingly, transfer of Ag-specific iNrp-1+CD4+ conv T cells aggravated diabetes development, whereas Ag-specific nNrp-1+CD4+ conv T cells failed to induce disease in a T cell transfer model of diabetes. Overall, our results indicate that Nrp-1 expression has opposite functions in recently activated CD4+ non-Tregs compared with CD4+ non-Tregs from naive mice.

Human Cord Blood B Cells Differ from the Adult Counterpart by Conserved Ig Repertoires and Accelerated Response Dynamics.

Neonatal and infant immune responses are characterized by a limited capability to generate protective Ab titers and memory B cells as seen in adults. Multiple studies support an immature or even impaired character of umbilical cord blood (UCB) B cells themselves. In this study, we provide a comprehensive molecular and functional comparison of B cell subsets from UCB and adult peripheral blood. Most UCB B cells have a mature, naive B cell phenotype as seen in adults. The UCB Ig repertoire is highly variable but interindividually conserved, as BCR clonotypes are frequently shared between neonates. Furthermore, UCB B cells show a distinct transcriptional program that confers accelerated responsiveness to stimulation and facilitated IgA class switching. Stimulation drives extensive differentiation into Ab-secreting cells, presumably limiting memory B cell formation. Humanized mice suggest that the distinctness of UCB versus adult B cells is already reflected by the developmental program of hematopoietic precursors, arguing for a layered B-1/B-2 lineage system as in mice, albeit our findings suggest only partial comparability to murine B-1 cells. Our study shows that UCB B cells are not immature or impaired but differ from their adult mature counterpart in a conserved BCR repertoire, efficient IgA class switching, and accelerated, likely transient response dynamics.

Regulatory T Cells Contribute to Sexual Dimorphism in Neonatal Hypoxic-Ischemic Brain Injury.

BACKGROUND AND PURPOSE: Neonatal encephalopathy caused by hypoxia-ischemia (HI) is a major cause of death and disability in newborns. Clinical and experimental studies suggest a sexual dimorphism in HI-induced brain injury and therapy responses. A major hallmark of HI pathophysiology is the infiltration of peripheral immune cells into the injured brain. However, the specific role of regulatory T cells (Tregs) in neonatal HI is still unknown. METHODS: Nine-day-old mice were exposed to HI by ligation of the right common carotid artery followed by 1 hour hypoxia (10% oxygen). Using immunohistochemistry, flow cytometry, and microarray analyses, Tregs were investigated in the brain, spleen, and blood 24 hours post HI. The functional role of Tregs was evaluated by acute Treg depletion in depletion of regulatory T cells transgenic mice. Brain injury, neuroinflammatory responses, and vascular injury were analyzed via immunohistochemistry and Western blot 48 hours and 7 days after HI. Functional outcome was assessed 3 days and 5 weeks after HI. RESULTS: Female mice revealed an increased cerebral Treg infiltration, coinciding with elevated chemokine receptor expression. Treg depletion in females aggravated HI-induced brain tissue injury, short-term motor deficits, and long-term deficits in exploratory activity, paralleled by an increased microglia and endothelial activation and leukocyte infiltration. Treg depletion in male mice reduced HI-induced brain injury, short-term motor, and long-term cognitive deficits, associated with reduced vascular injury. Ex vivo isolated female Tregs displayed an increased immunosuppressive activity on effector T cell proliferation and an increased gene enrichment in pathways related to enhanced Treg activity. CONCLUSIONS: Tregs from neonatal female mice provide endogenous neuroprotection, whereas Tregs from male mice increase secondary neurodegeneration. As potential mechanisms, we identified intrinsic transcriptional differences associated with enhanced anti-inflammatory activity of female Tregs. Our study emphasizes the urgent need for sex-stratified clinical and preclinical analyses.

A detailed analysis of F-MuLV- and SFFV-infected cells in Friend virus-infected mice reveals the contribution of both F-MuLV- and SFFV-infected cells to the interleukin-10 host response.

BACKGROUND: Friend virus (FV) is a complex of the Friend murine leukemia virus (F-MuLV) and the replication-defective, pathogenic spleen focus forming virus (SFFV). In the past, we used a fluorescently labeled F-MuLV to analyze FV target cells. To build on these findings, we have now created a double-labeled FV that contains a Katushka-labeled F-MuLV and an mTagBFP-labeled SFFV, which we have used to study the infection by the two individual viruses in the FV infection of highly susceptible BALB/c mice. RESULTS: Our data show that the target cells of SFFV largely mirror those of F-MuLV, with the highest virus loads in erythroblasts, B cells and myeloid cells. The early phase of infection was dominated by cells infected by either SFFV or F-MuLV, whereas double-infected cells became dominant later in the course of infection with increasing viral loads. In the late phase of infection, the frequency of double-infected cells was similarly high as the frequencies of SFFV or F-MuLV single-infected cells, and single- and double-infected cells outnumbered the uninfected cells in the most highly infected cell populations such as erythroblasts. FV and retroviruses in general have been shown to induce interleukin 10 (IL-10) as a means of suppressing immune responses. Interestingly, we found in infected IL-10-eGFP reporter mice that SFFV-infected cells contributed to the IL-10-producing cell pool much more significantly than F-MuLV-infected cells, suggesting that the truncated SFFV envelope protein gp55 might play a role in IL-10 induction. Even though BALB/c mice mount notoriously weak immune responses against FV, infection of mice with an ablation of IL-10 expression in T cells showed transiently lower viral loads and stronger T cell activation, suggesting that IL-10 induction by FV and by SFFV in particular may contribute to a suppressed immune response in BALB/c mice. CONCLUSION: Our data provide detailed information about both F-MuLV- and SFFV-infected cells during the course of FV infection in highly susceptible mice and imply that the pathogenic SFFV contributes to immune suppression.

CRISPR/Cas9-mediated demethylation of FOXP3-TSDR toward Treg-characteristic programming of Jurkat T cells.

Demethylation of FOXP3-TSDR (Treg specific demethylated region) is a hallmark of stable differentiation and suppressive function of regulatory T (Treg) cells. Previous protocols aiming at human naïve T cell differentiation failed to implement a Treg cell specific epigenetic signature. Ten-eleven translocation (TET) enzymes catalyze DNA demethylation. Plasmids towardexpression of a fusion protein encompassing nonfunctional Cas9, the catalytic domain of TET1, blue fluorescent protein, and encoding single guide RNAs (sgRNAs) targeting specific segments of the FOXP3-TSDR were engineered and transfected into Jurkat T cells. FOXP3-TSDR methylation was analyzed by deep-amplicon bisulfite sequencing while cellular Foxp3, Tbet, Gata3, and Rorgt mRNA levels were determined by real-time PCR. Overexpression of dCas9TET1 significantly decreased Jurkat cell FOXP3-TSDR methylation and increased Foxp3 mRNA expression while expressions of master transcription factor mRNAs of other major T cell lineages remained largely unaffected. dCas9-TET1 construct transfection mediated Treg programming of patients' primary T cells might be feasible.

Endogenous CD83 Expression in CD4+ Conventional T Cells Controls Inflammatory Immune Responses.

The glycoprotein CD83 is known to be expressed by different immune cells including activated CD4+Foxp3+ regulatory T cells (Tregs) and CD4+Foxp3- conventional T cells. However, the physiological function of endogenous CD83 in CD4+ T cell subsets is still unclear. In this study, we have generated a new CD83flox mouse line on BALB/c background, allowing for specific ablation of CD83 in T cells upon breeding with CD4-cre mice. Tregs from CD83flox/flox/CD4-cretg/wt mice had similar suppressive activity as Tregs from CD83flox/flox/CD4-crewt/wt wild-type littermates, suggesting that endogenous CD83 expression is dispensable for the inhibitory capacity of Tregs. However, CD83-deficient CD4+ conventional T cells showed elevated proliferation and IFN-γ secretion as well as an enhanced capacity to differentiate into Th1 cells and Th17 cells upon stimulation in vitro. T cell-specific ablation of CD83 expression resulted in aggravated contact hypersensitivity reaction accompanied by enhanced CD4+ T cell activation. Moreover, adoptive transfer of CD4+CD45RBhigh T cells from CD83flox/flox/CD4-cretg /wt mice into Rag2-deficient mice elicited more severe colitis associated with increased serum concentrations of IL-12 and elevated CD40 expression on CD11c+ dendritic cells (DCs). Strikingly, DCs from BALB/c mice cocultured with CD83-deficient CD4+ conventional T cells showed enhanced CD40 expression and IL-12 secretion compared with DCs cocultured with CD4+ conventional T cells from CD83flox/flox/CD4-crewt/wt wild-type mice. In summary, these results indicate that endogenous CD83 expression in CD4+ conventional T cells plays a crucial role in controlling CD4+ T cell responses, at least in part, by regulating the activity of CD11c+ DCs.

Heroin-assisted treatment of heroin-addicted patients normalizes regulatory T cells but does not restore CD4+ T cell proliferation.

Heroin dependence may result in suppression of adaptive immune responses. Previously, we demonstrated an increase in relative numbers of inhibitory CD4+ regulatory T cells (Tregs) and impaired proliferative activity of CD4+ T cells from heroin-addicted patients in contrast to patients in opioid maintenance therapy and healthy controls. However, it remains elusive whether heroin has a direct impact on the CD4+ T cell compartment or whether observed effects result from stressful living conditions. Here, we analyzed the frequencies of Tregs and the proliferation as well as the cytokine production of stimulated CD4+ T cells from heroin-addicted patients with use of illicit heroin, patients in heroin-assisted treatment (HAT), and patients in methadone maintenance therapy (MMT). Relative numbers of CD4+ Tregs were significantly enhanced in patients with illicit heroin abuse compared with patients in HAT or MMT. Notably, CD4+ T cells from patients in HAT and from persons using illicit heroin showed significant reduced proliferation and secretion of the pro-inflammatory cytokines IFN-γ and IL-6 upon stimulation in vitro. From these results, we conclude that structured programs such as HAT and MMT dampen elevated frequencies of Tregs in heroin-addicted patients, whereas chronic heroin administration irrespective of using illicit heroin or receiving HAT has a direct impact on the proliferative activity and cytokine production of CD4+ T cells.

Intestinal helminth infection drives carcinogenesis in colitis-associated colon cancer.

Inflammatory bowel diseases (IBD) are chronic inflammatory disorders of the gastrointestinal tract, strongly associated with an increased risk of colorectal cancer development. Parasitic infections caused by helminths have been shown to modulate the host's immune response by releasing immunomodulatory molecules and inducing regulatory T cells (Tregs). This immunosuppressive state provoked in the host has been considered as a novel and promising approach to treat IBD patients and alleviate acute intestinal inflammation. On the contrary, specific parasite infections are well known to be directly linked to carcinogenesis. Whether a helminth infection interferes with the development of colitis-associated colon cancer (CAC) is not yet known. In the present study, we demonstrate that the treatment of mice with the intestinal helminth Heligmosomoides polygyrus at the onset of tumor progression in a mouse model of CAC does not alter tumor growth and distribution. In contrast, H. polygyrus infection in the early inflammatory phase of CAC strengthens the inflammatory response and significantly boosts tumor development. Here, H. polygyrus infection was accompanied by long-lasting alterations in the colonic immune cell compartment, with reduced frequencies of colonic CD8+ effector T cells. Moreover, H. polygyrus infection in the course of dextran sulfate sodium (DSS) mediated colitis significantly exacerbates intestinal inflammation by amplifying the release of colonic IL-6 and CXCL1. Thus, our findings indicate that the therapeutic application of helminths during CAC might have tumor-promoting effects and therefore should be well-considered.

MicroRNA-183 and microRNA-96 are associated with autoimmune responses by regulating T cell activation.

MircoRNAs (miRs) are small molecules that regulate gene expression at the posttranscriptional level. They have been proposed to be involved in the regulation of several immune responses including autoimmunity. Here, we identified miR-183 and miR-96 to be highly expressed in CD4+ T cells from peripheral blood of Graves' orbitopathy (GO) patients as well as in human and murine T cells upon activation in vitro. By using Luciferase-based binding assays, we identified EGR-1 as target for miR-183 and miR-96. Overexpression of miR-183 and miR-96 in murine CD4+ T cells by retroviral gene transfer resulted in decreased EGR-1 and PTEN expression, elevated Akt phosphorylation and enhanced proliferation. In contrast, treatment of murine CD4+ T cells with specific antagomiRs increased EGR-1 and PTEN expression and interfered with the proliferative activity upon stimulation in vitro. Strikingly, adoptive transfer of miR-183 and miR-96 overexpressing antigen-specific T cells into INS-HA/Rag2KO mice accelerated the development of autoimmune diabetes, whereas transfer of antagomiR-treated cells delayed the disease onset. These results indicate that miR-183 and miR-96 have the ability to regulate the strength of T cell activation and thereby the development and severity of T cell-dependent autoimmune diseases.

Comparison of genotyping methods for Cunninghamella bertholletiae.

BACKGROUND: Invasive fungal infections caused by filamentous fungi of the order Mucorales are serious complications in immunocompromised patients and often associated with fatal outcome. As a member of this order, Cunninghamella bertholletiae is a saprophytic fungus with naturally exhibited high minimum inhibitory concentrations against common antifungal drugs and with the potential for outbreaks in clinical settings. OBJECTIVES AND METHODS: In a proof-of-principle study, we evaluated the performance of microsatellite markers for the discrimination of thirteen C. bertholletiae isolates from various sources in comparison with a repetitive sequence-based PCR (rep-PCR) and random amplification of polymorphic DNA (RAPD). Based on the higher discriminatory power of the microsatellite PCR with five separate primer pairs (Simpson's index of 1 vs 0 [RAPD] and 0 [rep-PCR]), the novel method was applied to eight additional isolates, including four well-characterised isolates from a cluster of infections in a next step. RESULTS: In total, microsatellite PCR identified 21 separate genotypes. A probable epidemiological association of the cluster isolates could be demonstrated by microsatellite genotyping. CONCLUSION: In conclusion, our findings demonstrate the value of microsatellite PCR in genotyping Cunninghamella bertholletiae and its potential for future applications with other species of the order Mucorales.

Interaction between hypothermia and delayed mesenchymal stem cell therapy in neonatal hypoxic-ischemic brain injury.

Acute hypothermia treatment (HT) is the only clinically established intervention following neonatal hypoxic-ischemic brain injury. However, almost half of all cooled infants still die or suffer from long-lasting neurological impairments. Regenerative therapies, such as mesenchymal stem cells (MSC) appear promising as adjuvant therapy. In the present study, we hypothesized that HT combined with delayed MSC therapy results in augmented protection, improving long-term neurological outcome. Postnatal day 9 (P9) C57BL/6 mice were exposed to hypoxia-ischemia followed by 4 h HT. Murine bone marrow-derived MSC (1 × 106 cells/animal) were administered intranasally at P12. Cytokine and growth factor levels were assessed by ELISA and Luminex® multiplex assay 24 h following MSC delivery. One week after HI, tissue injury and neuroinflammatory responses were determined by immunohistochemistry and western blot. Long-term motor-cognitive outcome was assessed 5 weeks post injury. MSC responses to the brains' environment were evaluated by gene expression analysis in MSC, co-cultured with brain homogenates isolated at P12. Both, MSC and HT improved motor deficits, while cognitive function could only be restored by MSC. Compared to each single therapy, combined treatment led to increased long-lasting motor-cognitive deficits and exacerbated brain injury, accompanied by enhanced endothelial activation and peripheral immune cell infiltration. MSC co-cultured with brain extracts of HT-treated animals revealed increased pro-inflammatory cytokine and decreased growth factor expression. In vivo protein analysis showed higher pro-inflammatory cytokine levels after combined treatment compared to single therapy. Furthermore, HI-induced increase in growth factors was normalized to control levels by HT and MSC single therapy, while the combination induced a further decline below control levels. Our results suggest that alteration of the brains' microenvironment by acute HT modulates MSC function resulting in a pro-inflammatory environment combined with alteration of the homeostatic growth factor milieu in the neonatal hypoxic-ischemic brain. This study delineates potential unexpected side effects of cell-based therapies as add-on therapy for acute hypothermia treatment.

TrkB-Target Galectin-1 Impairs Immune Activation and Radiation Responses in Neuroblastoma: Implications for Tumour Therapy.

Galectin-1 (Gal-1) has been described to promote tumour growth by inducing angiogenesis and to contribute to the tumour immune escape. We had previously identified up-regulation of Gal-1 in preclinical models of aggressive neuroblastoma (NB), the most common extracranial tumour of childhood. While Gal-1 did not confer a survival advantage in the absence of exogenous stressors, Gal-1 contributed to enhanced cell migratory and invasive properties. Here, we review these findings and extend them by analyzing Gal-1 mediated effects on immune cell regulation and radiation resistance. In line with previous results, cell autonomous effects as well as paracrine functions contribute to Gal-1 mediated pro-tumourigenic functions. Interfering with Gal-1 functions in vivo will add to a better understanding of the role of the Gal-1 axis in the complex tumour-host interaction during immune-, chemo- and radiotherapy of neuroblastoma.

Hypoxia Enhances Immunosuppression by Inhibiting CD4+ Effector T Cell Function and Promoting Treg Activity.

BACKGROUND/AIMS: Hypoxia occurs in many pathological conditions, including inflammation and cancer. Within this context, hypoxia was shown to inhibit but also to promote T cell responses. Due to this controversial function, we aimed to explore whether an insufficient anti-tumour response during colitis-associated colon cancer could be ascribed to a hypoxic microenvironment. METHODS: Colitis-associated colon cancer was induced in wildtype mice, and hypoxia as well as T cell immunity were analysed in the colonic tumour tissues. In addition, CD4+ effector T cells and regulatory T cells were cultured under normoxic and hypoxic conditions and examined regarding their phenotype and function. RESULTS: We observed severe hypoxia in the colon of mice suffering from colitis-associated colon cancer that was accompanied by a reduced differentiation of CD4+ effector T cells and an enhanced number and suppressive activity of regulatory T cells. Complementary ex vivo and in vitro studies revealed that T cell stimulation under hypoxic conditions inhibited the differentiation, proliferation and IFN-γ production of TH1 cells and enhanced the suppressive capacity of regulatory T cells. Moreover, we identified an active role for HIF-1α in the modulation of CD4+ T cell functions under hypoxic conditions. CONCLUSION: Our data indicate that oxygen availability can function as a local modulator of CD4+ T cell responses and thus influences tumour immune surveillance in inflammation-associated colon cancer.

Local delivery of siRNA-loaded calcium phosphate nanoparticles abates pulmonary inflammation.

The local interference of cytokine signaling mediated by siRNA-loaded nanoparticles might be a promising new therapeutic approach to dampen inflammation during pulmonary diseases. For the local therapeutic treatment of pulmonary inflammation, we produced multi-shell nanoparticles consisting of a calcium phosphate core, coated with siRNAs directed against pro-inflammatory mediators, encapsulated into poly(lactic-co-glycolic acid), and coated with a final outer layer of polyethylenimine. Nasal instillation of nanoparticles loaded with a mixture of siRNAs directed against different cytokines to mice suffering from TH1 cell-mediated lung inflammation, or of siRNA directed against NS-1 in an influenza infection model led to a significant reduction of target gene expression which was accompanied by distinct amelioration of lung inflammation in both models. Thus, this study provides strong evidence that the specific and local modulation of the inflammatory response by CaP/PLGA nanoparticle-mediated siRNA delivery could be a promising approach for the treatment of inflammatory disorders of the lung.

Plasmodium yoelii infection of BALB/c mice results in expansion rather than induction of CD4(+) Foxp3(+) regulatory T cells.

Recently, we demonstrated elevated numbers of CD4(+) Foxp3(+) regulatory T (Treg) cells in Plasmodium yoelii-infected mice contributing to the regulation of anti-malarial immune response. However, it remains unclear whether this increase in Treg cells is due to thymus-derived Treg cell expansion or induction of Treg cells in the periphery. Here, we show that the frequency of Foxp3(+) Treg cells expressing neuropilin-1 (Nrp-1) decreased at early time-points during P. yoelii infection, whereas percentages of Helios(+) Foxp3(+) Treg cells remained unchanged. Both Foxp3(+) Nrp-1(+) and Foxp3(+) Nrp-1(-) Treg cells from P. yoelii-infected mice exhibited a similar T-cell receptor Vß chain usage and methylation pattern in the Treg-specific demethylation region within the foxp3 locus. Strikingly, we did not observe induction of Foxp3 expression in Foxp3(-) T cells adoptively transferred to P. yoelii-infected mice. Hence, our results suggest that P. yoelii infection triggered expansion of naturally occurring Treg cells rather than de novo induction of Foxp3(+) Treg cells.

Combination of nanoparticle-based therapeutic vaccination and transient ablation of regulatory T cells enhances anti-viral immunity during chronic retroviral infection.

BACKGROUND: Regulatory T cells (Tregs) have been shown to limit anti-viral immunity during chronic retroviral infection and to restrict vaccine-induced T cell responses. The objective of the study was to assess whether a combinational therapy of nanoparticle-based therapeutic vaccination and concomitant transient ablation of Tregs augments anti-viral immunity and improves virus control in chronically retrovirus-infected mice. Therefore, chronically Friend retrovirus (FV)-infected mice were immunized with calcium phosphate (CaP) nanoparticles functionalized with TLR9 ligand CpG and CD8(+) or CD4(+) T cell epitope peptides (GagL85-93 or Env gp70123-141) of FV. In addition, Tregs were ablated during the immunization process. Reactivation of CD4(+) and CD8(+) effector T cells was analysed and the viral loads were determined. RESULTS: Therapeutic vaccination of chronically FV-infected mice with functionalized CaP nanoparticles transiently reactivated cytotoxic CD8(+) T cells and significantly reduced the viral loads. Transient ablation of Tregs during nanoparticle-based therapeutic vaccination strongly enhanced anti-viral immunity and further decreased viral burden. CONCLUSION: Our data illustrate a crucial role for CD4(+) Foxp3(+) Tregs in the suppression of anti-viral T cell responses during therapeutic vaccination against chronic retroviral infection. Thus, the combination of transient Treg ablation and therapeutic nanoparticle-based vaccination confers robust and sustained anti-viral immunity.