Regulatory Phenotype Research Articles

Liposome-based nanoparticles impact on regulatory and effector phenotypes of macrophages and T cells in multiple Sclerosis patients

Current available treatments of Multiple Sclerosis (MS) reduce neuroinflammation acting on different targets on the immune system, but potentially lead to severe side effects and have a limited efficacy in slowing the progression of the disease. Here, we evaluated in vitro the immunomodulatory potential of a new class of nanoparticles – liposomes, constituted by a double-layer of phosphatidylserine (PSCho/PS), and double-faced, with an outer layer of phosphatidylserine and an inner layer of phosphatidic acid (PSCho/PA), either alone or in the presence of the myelin basic protein (MBP) peptide (residues 85–99) (PSCho/PS-MBP and PSCho/PA-MBP). Results showed that PSCho/PS are equally and efficiently internalized by pro- and anti-inflammatory macrophages (M1 and M2 respectively), while PSCho/PA were internalized better by M2 than M1. PSCho/PS liposomes were able to inhibit the secretion of innate pro-inflammatory cytokine IL-1β. PSCho/PS liposomes expanded Tregs, reducing Th1 and Th17 cells, while PSCho/PA liposomes were unable to dampen pro-inflammatory T cells and to promote immune-regulatory phenotype (Treg). The ability of PSCho/PS liposomes to up-regulate Treg cells was more pronounced in MS patients with high basal expression of M2 markers. PSCho/PS liposomes were more effective in decreasing Th1 (but not Th17) cells in MS patients with a disease duration >3 months. On the other hand, down-modulation of Th17 cells was evident in MS patients with active, Gadolinium enhancing lesions at MRI and in MS patients with a high basal expression of M1-associated markers in the monocytes. The same findings were observed for the modulation of MBP-driven Th1/Th17/Treg responses.These observations suggest that early MS associate to a hard-wired pro-Th1 phenotype of M1 that is lost later during disease course. On the other hand, acute inflammatory events reflect a temporary decrease of M2 phenotype that however is amenable to restauration upon treatment with PSCho/PS liposomes. Thus, together these data indicate that monocytes/macrophages may play an important regulatory function during MS course and suggest a role for PSCho/PS and PSCho/PS-MBP as new therapeutic tools to dampen the pro-inflammatory immune responses and to promote its regulatory branch.

Impact of dietary vitamin D on immunoregulation and disease pathology in lupus-prone NZB/W F1 mice.

Vitamin D (VD) deficiency is a highly prevalent worldwide phenomenon and is extensively discussed as a risk factor for the development of systemic lupus erythematosus (SLE) and other immune-mediated diseases. In addition, it is now appreciated that VD possesses multiple immunomodulatory effects. This study aims to explore the impact of dietary VD intake on lupus manifestation and pathology in lupus-prone NZB/W F1 mice and identify the underlying immunological mechanisms modulated by VD. Here, we show that low VD intake accelerates lupus progression, reflected in reduced overall survival and an earlier onset of proteinuria, as well higher concentrations of anti-double-stranded DNA autoantibodies. This unfavorable effect gained statistical significance with additional low maternal VD intake during the prenatal period. Among examined immunological effects, we found that low VD intake consistently hampered the adoption of a regulatory phenotype in lymphocytes, significantly reducing both IL-10-expressing and regulatory CD4+ T cells. This goes along with a mildly decreased frequency of IL-10-expressing B cells. We did not observe consistent effects on the phenotype and function of innate immune cells, including cytokine production, costimulatory molecule expression, and phagocytic capacity. Hence, our study reveals that low VD intake promotes lupus pathology, likely via the deviation of adaptive immunity, and suggests that the correction of VD deficiency might not only exert beneficial functions by preventing osteoporosis but also serve as an important module in prophylaxis and as an add-on in the treatment of lupus and possibly other immune-mediated diseases. Further research is required to determine the most appropriate dosage, as too-high VD serum levels may also induce adverse effects, possibly also on lupus pathology.

Nasal administration of anti-CD3 monoclonal antibody modulates effector CD8+ T cell function and induces a regulatory response in T cells in human subjects.

Parenteral anti-CD3 Mab (OKT3) has been used to treat transplant rejection and parental administration of a humanized anti-CD3 Mab (Teplizumab) showed positive effects in diabetes. Nasal administration of anti-CD3 Mab has not been carried out in humans. Nasal anti-CD3 Mab suppresses autoimmune diseases and central nervous system (CNS) inflammation in animal models. We investigated the safety and immune effects of a fully humanized, previously uncharacterized nasal anti-CD3 Mab (Foralumab) in humans and its in vitro stimulatory properties. In vitro, Foralumab were compared to UCHT1 anti-human CD3 mAb. For human administration, 27 healthy volunteers (9 per group) received nasal Foralumab or placebo at a dose of 10ug, 50ug, or 250ug daily for 5 days. Safety was assessed and immune parameters measured on day 1 (pre-treatment), 7, 14, and 30 by FACS and by scRNAseq. In vitro, Foralumab preferentially induced CD8+ T cell stimulation, reduced CD4+ T cell proliferation and lowered expression of IFNg, IL-17 and TNFa. Foralumab induced LAP, TIGIT, and KLRG1 immune checkpoint molecules on CD8+ and CD4+ T cells in a mechanism independent of CD8 T cells. In vivo, nasal Foralumab did not modulate CD3 from the T cell surface at any dose. Immune effects were primarily observed at the 50ug dose and consisted of reduction of CD8+ effector memory cells, an increase in naive CD8+ and CD4+ T cells, and reduced CD8+ T cell granzyme B and perforin expression. Differentially expressed genes observed by scRNAseq in CD8+ and CD4+ populations promoted survival and were anti-inflammatory. In the CD8+ TEMRA population there was induction of TIGIT, TGFB1 and KIR3DL2, indicative of a regulatory phenotype. In the memory CD4+ population, there was induction of CTLA4, KLRG1, and TGFB whereas there was an induction of TGF-B1 in naïve CD4+ T cells. In monocytes, there was induction of genes (HLA-DP, HLA-DQ) that promote a less inflammatory immune response. No side effects were observed, and no subjects developed human anti-mouse antibodies. These findings demonstrate that nasal Foralumab is safe and immunologically active in humans and presents a new avenue for the treatment of autoimmune and CNS diseases.

Heterogeneity of neutrophils and inflammatory responses in patients with COVID-19 and healthy controls.

Severe respiratory viral infections, including SARS-CoV-2, have resulted in high mortality rates despite corticosteroids and other immunomodulatory therapies. Despite recognition of the pathogenic role of neutrophils, in-depth analyses of this cell population have been limited, due to technical challenges of working with neutrophils. We undertook an unbiased, detailed analysis of neutrophil responses in adult patients with COVID-19 and healthy controls, to determine whether distinct neutrophil phenotypes could be identified during infections compared to the healthy state. Single-cell RNA sequencing analysis of peripheral blood neutrophils from hospitalized patients with mild or severe COVID-19 disease and healthy controls revealed distinct mature neutrophil subpopulations, with relative proportions linked to disease severity. Disruption of predicted cell-cell interactions, activated oxidative phosphorylation genes, and downregulated antiviral and host defense pathway genes were observed in neutrophils obtained during severe compared to mild infections. Our findings suggest that during severe infections, there is a loss of normal regulatory neutrophil phenotypes seen in healthy subjects, coupled with the dropout of appropriate cellular interactions. Given that neutrophils are the most abundant circulating leukocytes with highly pathogenic potential, current immunotherapies for severe infections may be optimized by determining whether they aid in restoring an appropriate balance of neutrophil subpopulations.

Immuno(T)herapy for age-related diseases.

During the last decade, the stimulation of T-cell function by the blockage of immunosuppressive checkpoints has experienced an outstanding impact in the treatment of cancer. Development of the chimeric antigen receptor T-cell technology has also emerged as a powerful alternative for patients suffering from oncological processes, especially those affected by hematological neoplasms. Recent evidence suggest that the use of immunotherapy could be extended to non-oncological diseases and could be especially relevant for age-associated disorders, opening exciting therapeutic options for a wide range of diseases of the elderly. Here we comment on the emergence of T-cell-based immunotherapies as feasible approaches that could revolutionize the future of GeroScience.

Cell-cell contacts via N-cadherin induce a regulatory renin secretory phenotype in As4.1 cells.

The lack of a clonal renin-secreting cell line has greatly hindered the investigation of the regulatory mechanisms of renin secretion at the cellular, biochemical, and molecular levels. In the present study, we investigated whether it was possible to induce phenotypic switching of the renin-expressing clonal cell line As4.1 from constitutive inactive renin secretion to regulated active renin secretion. When grown to postconfluence for at least two days in media containing fetal bovine serum or insulin-like growth factor-1, the formation of cell-cell contacts via N-cadherin triggered downstream cellular signaling cascades and activated smooth muscle-specific genes, culminating in phenotypic switching to a regulated active renin secretion phenotype, including responding to the key stimuli of active renin secretion. With the use of phenotype-switched As4.1 cells, we provide the first evidence that active renin secretion via exocytosis is regulated by phosphorylation/dephosphorylation of the 20 kDa myosin light chain. The molecular mechanism of phenotypic switching in As4.1 cells described here could serve as a working model for full phenotypic modulation of other secretory cell lines with incomplete phenotypes.

FoxP3 expression by retinal pigment epithelial cells: transcription factor with potential relevance for the pathology of age-related macular degeneration

BackgroundForkhead-Box-Protein P3 (FoxP3) is a transcription factor and marker of regulatory T cells, converting naive T cells into Tregs that can downregulate the effector function of other T cells. We previously detected the expression of FoxP3 in retinal pigment epithelial (RPE) cells, forming the outer blood–retina barrier of the immune privileged eye.MethodsWe investigated the expression, subcellular localization, and phosphorylation of FoxP3 in RPE cells in vivo and in vitro after treatment with various stressors including age, retinal laser burn, autoimmune inflammation, exposure to cigarette smoke, in addition of IL-1β and mechanical cell monolayer destruction. Eye tissue from humans, mouse models of retinal degeneration and rats, and ARPE-19, a human RPE cell line for in vitro experiments, underwent immunohistochemical, immunofluorescence staining, and PCR or immunoblot analysis to determine the intracellular localization and phosphorylation of FoxP3. Cytokine expression of stressed cultured RPE cells was investigated by multiplex bead analysis. Depletion of the FoxP3 gene was performed with CRISPR/Cas9 editing.ResultsRPE in vivo displayed increased nuclear FoxP3-expression with increases in age and inflammation, long-term exposure of mice to cigarette smoke, or after laser burn injury. The human RPE cell line ARPE-19 constitutively expressed nuclear FoxP3 under non-confluent culture conditions, representing a regulatory phenotype under chronic stress. Confluently grown cells expressed cytosolic FoxP3 that was translocated to the nucleus after treatment with IL-1β to imitate activated macrophages or after mechanical destruction of the monolayer. Moreover, with depletion of FoxP3, but not of a control gene, by CRISPR/Cas9 gene editing decreased stress resistance of RPE cells.ConclusionOur data suggest that FoxP3 is upregulated by age and under cellular stress and might be important for RPE function.

Stimulation of naïve B cells with a fusion protein consisting of FlaA and Bet v 1 induces regulatory B cells ex vivo.

The experimental fusion protein rFlaA:Betv1 was shown to efficiently suppress allergen-specific sensitization in mice. However, the detailed mechanism of rFlaA:Betv1-mediated immune modulation is not fully understood. In this study, we investigated the effect of rFlaA:Betv1 on naïve murine B cells. Immune modulating capacity of rFlaA:Betv1 was screened in IL-10 reporter mice. B cells were isolated from spleens of naïve C57Bl/6, TLR5-/- , or MyD88-/- mice, stimulated with rFlaA:Betv1 and controls, and monitored for the expression of the regulatory B cell markers CD1d, CD24, CD38, and surface IgM by flow cytometry. Secreted cytokines, antibodies, and reactivity of the induced antibodies were investigated by ELISA and intracellular flow cytometry. Suppressive capacity of rFlaA:Betv1-stimulated B cells was tested in mDC:CD4+ T cell:B cell triple cultures. Upon in vivo application of rFlaA:Betv1 into IL-10-GFP reporter mice, CD19+ B cells were shown to produce anti-inflammatory IL-10, suggesting B cells to contribute to the immune-modulatory properties of rFlaA:Betv1. rFlaA:Betv1-induced IL-10 secretion was confirmed in human B cells isolated from buffy coats. In vitro stimulation of naïve murine B cells with rFlaA:Betv1 resulted in an mTOR- and MyD88-dependent production of IL-10 and rFlaA:Betv1 induced Bet v 1-reactive IgG production, which was not observed for IgA. rFlaA:Betv1-stimulated B cells formed a CD19+ CD24+ CD1d+ IgM+ CD38+ Breg subpopulation capable of suppressing Bet v 1-induced TH2 cytokine secretion in vitro. rFlaA:Betv1 can act as a thymus-independent B cell antigen, stimulating the mTOR- and MyD88-dependent differentiation of B cells displaying a regulatory phenotype, IL-10 secretion, antigen-binding antibody production, and a suppressive capacity in vitro.

Development and function of regulatory innate lymphoid cells.

Innate lymphoid cells (ILCs) are a critical element of the innate immune system and are potent producers of pro-inflammatory cytokines. Recently, however, the production of the anti-inflammatory cytokine IL-10 has been observed in all ILC subtypes (ILC1s, ILC2s, and ILC3s) suggesting their ability to adopt a regulatory phenotype that serves to maintain lung and gut homeostasis. Other studies advocate a potential therapeutic role of these IL-10-expressing ILCs in allergic diseases such as asthma, colitis, and pancreatic islet allograft rejection. Herein, we review IL-10 producing ILCs, discussing their development, function, regulation, and immunotherapeutic potential through suppressing harmful inflammatory responses. Furthermore, we address inconsistencies in the literature regarding these regulatory IL-10 producing ILCs, as well as directions for future research.

NK cells are associated with immunometabolic response to a single exercise exertion in heart failure patients

Abstract Background Increased systemic inflammation and metabolic dysfunction are observed in heart failure with reduced ejection fraction (HFrEF). On the other hand, cardiorespiratory exercise testing (CPET) exerts a physical challenge and initiates the activation of the immune system, including acute release of natural killer (NK) cells into the circulation, and several metabolic pathways. Aim To characterize the inflammatory and metabolic alterations of HFrEF patients in response to an acute exercise challenge, and after 2 hours of recovery. Methods Participants with HFrEF (n=16), age and sex matched controls (CON, n=13) were investigated at baseline, immediately after and 2 hours after CPET. Clinical and physiological parameters, leukocyte profile, plasma cytokines and metabolites were assessed along with inflammatory and metabolic parameters at all three time points. NK cell counts and morphological/activation parameters in different contexts were examined. Further, the time-dependent coordination of NK cell numbers post-exercise with tryptophan metabolism and plasma triglycerides were assessed. NK cells were isolated from blood of healthy donors for ex vivo proof-of-principle experiments, including phenotype polarization and NK cell specific tryptophan metabolism. Results Cardiovascular risk profiles as well as leukocyte, cytokine and metabolic parameters at baseline were similar in CON and HFrEF. Immediately after CPET, lactate, and NK T cell blood counts were significantly increased in both groups. In HFrEF but not CON, platelet aggregates with NK cells, CD8+ cytotoxic T cells and “classical” CD14++CD16-monocytes, phosphatidylcholines and triglycerides were increased. After 2h of recovery, almost all altered parameters returned to baseline in CON. In contrast, blood counts and morphological markers of inflammatory effector cell types, including CD8+ T cells and neutrophils remained elevated in HFrEF. NK cells remained elevated after the recovery period and correlated with levels of various triglyceride species in the HFrEF patients. Tryptophan levels in plasma were reduced by acute exercise and the kynurenine to tryptophan ratio was increased and correlated with increase in NK and NK-T cell counts, as well as IL-12 plasma levels. Treatment with IL-12 led to increased synthesis of kynurenine from tryptophan, expression of indoleamine 2,3-dioxygenase and abundance of regulatory CD56bri NK cell phenotypes ex vivo. Secretome of untreated NK cells impaired cellular respiration, increased glycolysis/oxidation ratio in skeletal muscle cells, and increased the release of triglycerides from hepatocarcinoma cells. Conclusion CPET induced a complex acute immunometabolic response, whose restitution to baseline levels differed between HFrEF and healthy controls. Exercise-induced changes in NK cell metabolism and phenotype shift might modulate cellular respiration in myocytes and release of triglycerides by hepatocytes in HFrEF and in CON. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): DZHK (German Centre for Cardiovascular Research)

CD1d-independent NK1.1+ Treg cells are IL2-inducible Foxp3+ T cells co-expressing immunosuppressive and cytotoxic molecules.

Regulatory T cells (Treg) play pivotal roles in maintaining self-tolerance and preventing immunological diseases such as allergy and autoimmunity through their immunosuppressive properties. Although Treg cells are heterogeneous populations with distinct suppressive functions, expression of natural killer (NK) cell receptors (NKR) by these cells remains incompletely explored. Here we identified that a small population of Foxp3+CD4+ Treg cells in mice expresses the NK1.1 NKR. Furthermore, we found that rare NK1.1+ subpopulations among CD4+ Treg cells develop normally in the spleen but not the thymus through CD1d-independent pathways. Compared with NK1.1- conventional Treg cells, these NK1.1+ Treg cells express elevated Treg cell phenotypic hallmarks, pro-inflammatory cytokines, and NK cell-related cytolytic mediators. Our results suggest that NK1.1+ Treg cells are phenotypically hybrid cells sharing functional properties of both NK and Treg cells. Interestingly, NK1.1+ Treg cells preferentially expanded in response to recombinant IL2 stimulation in vitro, consistent with their increased IL2Rαβ expression. Moreover, DO11.10 T cell receptor transgenic NK1.1+ Treg cells were expanded in an ovalbumin antigen-specific manner. In the context of lipopolysaccharide-induced systemic inflammation, NK1.1+ Treg cells downregulated immunosuppressive molecules but upregulated TNFα production, indicating their plastic adaptation towards a more pro-inflammatory rather than regulatory phenotype. Collectively, we propose that NK1.1+ Treg cells might play a unique role in controlling inflammatory immune responses such as infection and autoimmunity.

Vaccination with a shared oncogenic tumor-self antigen elicits a population of CD8+ T cells with a regulatory phenotype

ABSTRACT Cancer immunotherapy is a powerful tool for inducing antigen-specific antitumor cytotoxic T lymphocytes (CTLs). Next-generation strategies may include vaccination against overexpressed oncogenic tumor-self antigens. Previously, we reported vaccination against the oncogenic tumor-self antigen D52 (D52) was effective in preventing tumor growth. We recently reported that D52-vaccinated IL-10-deficient mice generated a significant memory response against tumor recurrence compared to wild-type mice and that vaccine-induced CD8+ IL-10+ T cells may possess regulatory function. Herein, we extended these studies by testing the hypothesis that D52-vaccine-elicited CD8+ IL-10+ T cells represent a distinct T cell population with a regulatory phenotype. C57Black/6J mice deficient in IL-10 or IFN-γ were vaccinated with the murine orthologue of D52; vaccination of wild-type (wt) mice served as a control for comparison. T cells were isolated from all three groups of vaccinated mice, and RNA was extracted from purified CD8+ T cells for deep sequencing and expression analysis. Chemokine receptor 8 (CCR8) and inducible co-stimulator (ICOS) were overexpressed in CD8+ T cells that produced IL-10 but not IFN-γ. These surface markers are associated with IL-10 producing CD4+ T regulatory cells thus supporting the possibility that CD8+ IL-10+ T cells elicited by D52 vaccination represent a unique regulatory T cell subset. The current phenotypic analyses of D52 vaccine elicited CD8+ T cells strengthen our premise that CD8+ IL-10+ T cells elicited by D52 tumor-self protein vaccination likely contribute to the suppression of memory CTL responses and inhibition of durable tumor immunity.

Landscape and age dynamics of immune cells in the Egyptian rousette bat.

Bats harbor high-impact zoonotic viruses often in the absence of disease manifestation. This restriction and disease tolerance possibly rely on specific immunological features. In-depth molecular characterization of cellular immunity and imprinting of age on leukocyte compartments remained unexplored in bats. We employ single-cell RNA sequencing (scRNA-seq) and establish immunostaining panels to characterize the immune cell landscape in juvenile, subadult, and adult Egyptian rousette bats (ERBs). Transcriptomic and flow cytometry data reveal conserved subsets and substantial enrichments of CD79a+ B cells and CD11b+ Tcells in juvenile animals, whereas neutrophils, CD206+ myeloid cells, and CD3+ Tcells dominate as bats reach adulthood. Despite differing frequencies, phagocytosis of circulating and tissue-resident myeloid cells and proliferation of peripheral and splenic lymphocytes are analogous in juvenile and adult ERBs. We provide a comprehensive map of the immune landscape in ERBs and show age-imprinted resilience progression and find that variability in cellular immunity only partly recapitulates mammalian archetypes.

Recombinant pregnancy-specific glycoprotein-1-Fc reduces functional deficit in a mouse model of permanent brain ischaemia

BackgroundThe well-characterised role of the immune system in acute ischaemic stroke has prompted the search for immunomodulatory therapies. Pregnancy-specific glycoproteins (PSGs) are a group of proteins synthesised by placental trophoblasts which show immunomodulatory properties. The aim of this study was to determine whether a proposed PSG1-based therapeutic enhanced recovery in a mouse model of brain ischaemia and to explore possible immunomodulatory effects. MethodsMice underwent permanent electrocoagulation of the left middle cerebral artery (pMCAO). They received saline (n = 20) or recombinant pregnancy-specific glycoprotein-1-alpha “fused” to the Fc domain of IgG1 (rPSG1-Fc) (100 μg) (n = 22) at 1 h post-ischaemia. At 3 and 5 days post-ischaemia, neurobehavioural recovery was assessed by the grid-walking test. At 5 days post-ischaemia, lesion size was determined by NeuN staining. Peripheral T cell populations were quantified via flow cytometry. Immunohistochemistry was used to quantify ICAM-1 expression and FoxP3+ cell infiltration in the ischaemic brain. Immunofluorescence was employed to determine microglial activation status via Iba-1 staining.Results: rPSG1-Fc significantly enhanced performance in the grid-walking test at 3 and 5 days post-ischaemia. No effect on infarct size was observed. A significant increase in circulating CD4+ FoxP3+ cells and brain-infiltrating FoxP3+ cells was noted in rPSG1-Fc-treated mice. Among CD4+ cells, rPSG1-Fc enhanced the expression of IL-10 in spleen, blood, draining lymph nodes, and non-draining lymph nodes, while downregulating IFN-γ and IL-17 in spleen and blood. A similar cytokine expression pattern was observed in CD8+ cells. rPSG1-Fc reduced activated microglia in the infarct core. ConclusionThe administration of rPSG1-Fc improved functional recovery in post-ischaemic mice without impacting infarct size. Improved outcome was associated with a modulation of the cytokine-secreting phenotype of CD4+ and CD8+ T cells towards a more regulatory phenotype, as well as reduced activation of microglia. This establishes proof-of-concept of rPSG1-Fc as a potential stroke immunotherapy.

Secreted filarial nematode galectins modulate host immune cells.

Lymphatic filariasis (LF) is a mosquito-borne disease caused by filarial nematodes including Brugia malayi. Over 860 million people worldwide are infected or at risk of infection in 72 endemic countries. The absence of a protective vaccine means that current control strategies rely on mass drug administration programs that utilize inadequate drugs that cannot effectively kill adult parasites, thus established infections are incurable. Progress to address deficiencies in the approach to LF control is hindered by a poor mechanistic understanding of host-parasite interactions, including mechanisms of host immunomodulation by the parasite, a critical adaptation for establishing and maintaining infections. The canonical type 2 host response to helminth infection characterized by anti-inflammatory and regulatory immune phenotypes is modified by filarial nematodes during chronic LF. Current efforts at identifying parasite-derived factors driving this modification focus on parasite excretory-secretory products (ESP), including extracellular vesicles (EVs). We have previously profiled the cargo of B. malayi EVs and identified B. malayi galectin-1 and galectin-2 as among the most abundant EV proteins. In this study we further investigated the function of these proteins. Sequence analysis of the parasite galectins revealed highest homology to mammalian galectin-9 and functional characterization identified similar substrate affinities consistent with this designation. Immunological assays showed that Bma-LEC-2 is a bioactive protein that can polarize macrophages to an alternatively activated phenotype and selectively induce apoptosis in Th1 cells. Our data shows that an abundantly secreted parasite galectin is immunomodulatory and induces phenotypes consistent with the modified type 2 response characteristic of chronic LF infection.

Antigens from the parasitic nematode Trichuris suis induce metabolic reprogramming and trained immunity to constrain inflammatory responses in macrophages.

Regulation of macrophage (Mɸ) function can maintain tissue homeostasis and control inflammation. Parasitic worms (helminths) are potent modulators of host immune and inflammatory responses. They have evolved various strategies to promote immunosuppression, including redirecting phagocytic cells toward a regulatory phenotype. Although soluble products from the whipworm Trichuris suis (TSPs) have shown significant effects on Mɸ function, the mechanisms underlying these modulatory effects are still not well understood. In this study, we find that TSPs suppressed inflammatory cytokines (TNF and IL-6) in Mɸs stimulated with a broad panel of TLR agonists, whilst inducing IL-10. Moreover, M1 markers such as MHCII, CD86, iNOS, and TNF were downregulated in TSP-treated Mɸs, without polarizing them towards an M2-like phenotype. We showed that TSPs could establish a suppressed activation state of Mɸs lasting at least for 72h, indicating an anti-inflammatory innate training. Moreover, we found that TSPs, via repression of intracellular TNF generation, decreased its secretion rather than interfering with the release of surface-bound TNF. Metabolic analysis showed that TSPs promote oxidative phosphorylation (OXPHOS) without affecting glycolytic rate. Collectively, these findings expand our knowledge on helminth-induced immune modulation and support future investigations into the anti-inflammatory properties of TSPs for therapeutic purposes.

Enriched circulating and tumor-resident TGF-β+ regulatory B cells in patients with melanoma promote FOXP3+ Tregs

ABSTRACT B cells are emerging as key players of anti-tumor adaptive immune responses. We investigated regulatory and pro-inflammatory cytokine-expressing B cells in patients with melanoma by flow cytometric intracellular cytokine, CyTOF, transcriptomic, immunofluorescence, single-cell RNA-seq, and B:T cell co-culture analyses. We found enhanced circulating regulatory (TGF-β+ and PD-L1+) and reduced pro-inflammatory TNF-α+ B cell populations in patients compared with healthy volunteers (HVs), including lower IFN-γ+:IL-4+ and higher TGF-β+:TNF-α+ B cell ratios in patients. TGF-β-expressing B cells in the melanoma tumor microenvironment assembled in clusters and interacted with T cells via lymphoid recruitment (SELL, CXCL13, CCL4, CD74) signals and with Tregs via CD47:SIRP-γ, and FOXP3-promoting Galectin-9:CD44. While reduced in tumors compared to blood, TNF-α-expressing B cells engaged in crosstalk with Tregs via TNF-α signaling and the ICOS/ICOSL axis. Patient-derived B cells promoted FOXP3+ Treg differentiation in a TGF-β-dependent manner, while sustaining expression of IFN-γ and TNF-α by autologous T-helper cells and promoting T-helper cell proliferation ex vivo, an effect further enhanced with anti-PD-1 checkpoint blockade. Our findings reveal cytokine-expressing B cell compartments skewed toward regulatory phenotypes in patient circulation and melanoma lesions, intratumor spatial localization, and bidirectional crosstalk between B and T cell subsets with immunosuppressive attributes.

Bone Marrow-Derived Mesenchymal Stromal Cell Therapy in Severe COVID-19: Preliminary Results of a Phase I/II Clinical Trial

BackgroundTreatment of acute respiratory distress syndrome (ARDS) associated with COronaVIrus Disease-2019 (COVID-19) currently relies on dexamethasone and supportive mechanical ventilation, and remains associated with high mortality. Given their ability to limit inflammation, induce immune cells into a regulatory phenotype and stimulate tissue repair, mesenchymal stromal cells (MSCs) represent a promising therapy for severe and critical COVID-19 disease, which is associated with an uncontrolled immune-mediated inflammatory response.MethodsIn this phase I-II trial, we aimed to evaluate the safety and efficacy of 3 intravenous infusions of bone marrow (BM)-derived MSCs at 3-day intervals in patients with severe COVID-19. All patients also received dexamethasone and standard supportive therapy. Between June 2020 and September 2021, 8 intensive care unit patients requiring supplemental oxygen (high-flow nasal oxygen in 7 patients, invasive mechanical ventilation in 1 patient) were treated with BM-MSCs. We retrospectively compared the outcomes of these MSC-treated patients with those of 24 matched control patients. Groups were compared by paired statistical tests.ResultsMSC infusions were well tolerated, and no adverse effect related to MSC infusions were reported (one patient had an ischemic stroke related to aortic endocarditis). Overall, 3 patients required invasive mechanical ventilation, including one who required extracorporeal membrane oxygenation, but all patients ultimately had a favorable outcome. Survival was significantly higher in the MSC group, both at 28 and 60 days (100% vs 79.2%, p = 0.025 and 100% vs 70.8%, p = 0.0082, respectively), while no significant difference was observed in the need for mechanical ventilation nor in the number of invasive ventilation-free days, high flow nasal oxygenation-free days, oxygen support-free days and ICU-free days. MSC-treated patients also had a significantly lower day-7 D-dimer value compared to control patients (median 821.0 µg/L [IQR 362.0-1305.0] vs 3553 µg/L [IQR 1155.0-6433.5], p = 0.0085).ConclusionsBM-MSC therapy is safe and shows very promising efficacy in severe COVID-19, with a higher survival in our MSC cohort compared to matched control patients. These observations need to be confirmed in a randomized controlled trial designed to demonstrate the efficacy of BM-MSCs in COVID-19 ARDS.Clinical Trial Registration(www.ClinicalTrials.gov), identifier NCT04445454

The impact of HIV infection on the frequencies, function, spatial localization and heterogeneity of T follicular regulatory cells (TFRs) within human lymph nodes

BackgroundHIV eradication efforts have been unsuccessful partly due to virus persistence in immune sanctuary sites such as germinal centres within lymph node (LN) tissues. Recent evidence suggests that LNs harbour a novel subset of regulatory T cells, termed follicular regulatory T cells (TFRs), but their role in HIV pathogenesis is not fully elucidated.ResultsPaired excisional LN and peripheral blood samples obtained from 20 HIV-uninfected and 31 HIV-infected treated and 7 chronic untreated, were used to determine if and how HIV infection modulate frequencies, function and spatial localization of TFRs within LN tissues. Imaging studies showed that most TFRs are localized in extra-follicular regions. Co-culture assays showed TFRs suppression of TFH help to B cells. Importantly, epigenetic and transcriptional studies identified DPP4 and FCRL3 as novel phenotypic markers that define four functionally distinct TFR subpopulations in human LNs regardless of HIV status. Imaging studies confirmed the regulatory phenotype of DPP4+TFRs.ConclusionTogether these studies describe TFRs dynamic changes during HIV infection and reveal previously underappreciated TFR heterogeneity within human LNs.

Helios Expression Is Downregulated on CD8+ Treg in Two Mouse Models of Lupus During Disease Progression.

T-cell–mediated autoimmunity reflects an imbalance in this compartment that is not restored by tolerogenic immune cells, e.g., regulatory T cells or tolerogenic dendritic cells (tolDCs). Although studies into T-cell equilibrium have mainly focused on regulatory CD4+FoxP3+ T cells (CD4+ Tregs), recent findings on the lesser known CD8+ Tregs (CD44+CD122+Ly49+) have highlighted their non-redundant role in regulating lupus-like disease and their regulatory phenotype facilitated by the transcription factor Helios in mice and humans. However, there are still remaining questions about Helios regulation and dynamics in different autoimmune contexts. Here, we show the absence of CD8+ Tregs in two lupus-prone murine models: MRL/MPJ and MRL/lpr, in comparison with a non-prone mouse strain like C57BL/6. We observed that all MRL animals showed a dramatically reduced population of CD8+ Tregs and a greater Helios downregulation on diseased mice. Helios induction was detected preferentially on CD8+ T cells from OT-I mice co-cultured with tolDCs from C57BL/6 but not in MRL animals. Furthermore, the Helios profile was also altered in other relevant T-cell populations implicated in lupus, such as CD4+ Tregs, conventional CD4+, and double-negative T cells. Together, these findings could make Helios a versatile maker across the T-cell repertoire that is capable of differentiating lupus disease states.