The deficiency of DNASE1L3 does not affect systemic sclerosis pathogenesis in two inducible murine models of the disease.

We induced systemic sclerosis (SSc)-like disease in both wild-type and Dnase1l3-deficient mice using two distinct approaches involving bleomycin and hypochlorous acid injections. Our observations revealed that the deficiency in DNASE1L3 did not affect tissue fibrosis or inflammation caused by these treatments. Despite the association of single nucleotide polymorphisms in humans with SSc pathogenesis, our study demonstrates that DNASE1L3 is dispensable in two inducible murine models of SSc-like pathogenesis.

C-type lectin-like receptor LOX-1 promotes dendritic cell-mediated class-switched B cell responses.

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a pattern-recognition receptor for a variety of endogenous and exogenous ligands. However, LOX-1 function in the host immune response is not fully understood. Here, we report that LOX-1 expressed on dendritic cells (DCs) and B cells promotes humoral responses. On B cells LOX-1 signaling upregulated CCR7, promoting cellular migration toward lymphoid tissues. LOX-1 signaling on DCs licensed the cells to promote B cell differentiation into class-switched plasmablasts and led to downregulation of chemokine receptor CXCR5 and upregulation of chemokine receptor CCR10 on plasmablasts, enabling their exit from germinal centers and migration toward local mucosa and skin. Finally, we found that targeting influenza hemagglutinin 1 (HA1) subunit to LOX-1 elicited HA1-specific protective antibody responses in rhesus macaques. Thus, LOX-1 expressed on B cells and DC cells has complementary functions to promote humoral immune responses.

Cell type-specific expression of estrogen and progesterone receptors in the human vaginal mucosa.

Female sex hormones affect the immune response in the lower female genital tract. To understand their mechanisms of action, it is essential to define cell types expressing estrogen receptor (ER) and/or progesterone receptor (PR) in the human vaginal mucosa (VM). Here, we report that none of the dendritic cell (DC) subsets in the human VM expressed ERα or PR in situ. However, they were capable of expressing ERα, but not PR, after in vitro culture of the whole VM tissues. Similarly, ERα and/or PR expression by T cells in the VM tissues was also inducible rather than constitutive. In contrast, ERα and/or PR were constitutively expressed in HLA-DR- non-immune cell types (vimentin+, desmin+, or CD10+). These new findings will help us understand the mechanisms of action of female sex hormones in the modulation of immune response in the human VM and lower female genital tract.

Beyond the tumour microenvironment.

In contrast to the once dominant tumour-centric view of cancer, increasing attention is now being paid to the tumour microenvironment (TME), generally understood as the elements spatially located in the vicinity of the tumour. Thinking in terms of TME has proven extremely useful, in particular because it has helped identify and comprehend the role of nongenetic and noncell-intrinsic factors in cancer development. Yet some current approaches have led to a TME-centric view, which is no less problematic than the former tumour-centric vision of cancer, insofar as it tends to overlook the role of components located beyond the TME, in the 'tumour organismal environment' (TOE). In this minireview, we highlight the explanatory and therapeutic shortcomings of the TME-centric view and insist on the crucial importance of the TOE in cancer progression.

Increase of follicular helper T cells skewed toward a Th1 profile in CVID patients with non-infectious clinical complications.

Common variable immunodeficiency (CVID) is characterized by low levels of circulating immunoglobulins and defects in B cell maturation leading to an increased susceptibility to infections. Some patients develop complications such as autoimmune diseases, enteropathy, and lymphoproliferation, resulting in higher morbidity and mortality. Follicular helper T (Tfh) cells are specialized in helping B cell differentiation into Ig-producing cells. Three subsets have been described, namely non B-cell helper Tfh1 and the two B-helper cell subsets Tfh2 and Tfh17. We determined that circulating Tfh cells were elevated in CVID patients and skewed toward a Tfh1 profile. Interestingly, elevated levels of Tfh1 cells were significant only in patients harboring non-infectious complications regardless of the type of complication and inversely correlated with switched memory B cells. Moreover, CXCR3+ cells are increased in splenic CVID germinal centers. Our observations suggest that the altered balance in Tfh subsets in CVID is linked to a more severe disease.

The Antigen-Presenting Potential of Vγ9Vδ2 T Cells During Plasmodium falciparum Blood-Stage Infection.

During Plasmodium falciparum infections, erythrocyte-stage parasites inhibit dendritic cell maturation and function, compromising effective antimalarial adaptive immunity. Human Vγ9Vδ2 T cells can act in vitro as antigen-presenting cells (APCs) and induce αß T-cell activation. However, the relevance of this activity in vivo has remained elusive. Because Vγ9Vδ2 T cells are activated during the early immune response against P. falciparum infection, we investigated whether they could contribute to the instruction of adaptive immune responses toward malaria parasites. In P. falciparum-infected patients, Vγ9Vδ2 T cells presented increased surface expression of APC-associated markers HLA-DR and CD86. In response to infected red blood cells in vitro, Vγ9Vδ2 T cells upregulated surface expression of HLA-DR, HLA-ABC, CD40, CD80, CD83, and CD86, induced naive αß T-cell responses, and cross- presented soluble prototypical protein to antigen-specific CD8+ T cells. Our findings qualify Vγ9Vδ2 T cells as alternative APCs, which could be harnessed for therapeutic interventions and vaccine design.

Induction and activation of human Th17 by targeting antigens to dendritic cells via dectin-1.

Recent compelling evidence indicates that Th17 confer host immunity against a variety of microbes, including extracellular and intracellular pathogens. Therefore, understanding mechanisms for the induction and activation of Ag-specific Th17 is important for the rational design of vaccines against pathogens. To study this, we employed an in vitro system in which influenza hemagglutinin (HA) 1 was delivered to dendritic cells (DCs) via Dectin-1 using anti-human Dectin-1 (hDectin-1)-HA1 recombinant fusion proteins. We found that healthy individuals maintained broad ranges of HA1-specific memory Th17 that were efficiently activated by DCs targeted with anti-hDectin-1-HA1. Nonetheless, these DCs were not able to induce a significant level of HA1-specific Th17 responses even in the presence of the Th17-promoting cytokines IL-1ß and IL-6. We further found that the induction of surface IL-1R1 expression by signals via TCRs and common γ-chain receptors was essential for naive CD4(+) T cell differentiation into HA1-specific Th17. This process was dependent on MyD88, but not IL-1R-associated kinase 1/4. Thus, interruptions in STAT3 or MyD88 signaling led to substantially diminished HA1-specific Th17 induction. Taken together, the de novo generation of pathogen-specific human Th17 requires complex, but complementary, actions of multiple signals. Data from this study will help us design a new and effective vaccine strategy that can promote Th17-mediated immunity against microbial pathogens.

IL-26 is overexpressed in chronically HCV-infected patients and enhances TRAIL-mediated cytotoxicity and interferon production by human NK cells.

OBJECTIVE: Interleukin-26 (IL-26) is a member of the IL-10 cytokine family, first discovered based on its peculiar expression by virus-transformed T cells. IL-26 is overexpressed in chronic inflammation (rheumatoid arthritis and Crohn's disease) and induces proinflammatory cytokines by myeloid cells and some epithelial cells. We thus investigated the expression and potential role of IL-26 in chronic HCV infection, a pathology associated with chronic inflammation. DESIGN: IL-26 was quantified in a cohort of chronically HCV-infected patients, naive of treatment and its expression in the liver biopsies investigated by immunohistochemistry. We also analysed the ability of IL-26 to modulate the activity of natural killer (NK) cells, which control HCV infection. RESULTS: The serum levels of IL-26 are enhanced in chronically HCV-infected patients, mainly in those with severe liver inflammation. Immunohistochemistry reveals an intense IL-26 staining in liver lesions, mainly in infiltrating CD3+ cells. We also show that NK cells from healthy subjects and from HCV-infected patients are sensitive to IL-26. IL-26 upregulates membrane tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) expression on CD16- CD56(bright) NK cells, enabling them to kill HCV-infected hepatoma cells, with the same efficacy as interferon (IFN)-α-treated NK cells. IL-26 also induces the expression of the antiviral cytokines IFN-ß and IFN-γ, and of the proinflammatory cytokines IL-1ß and TNF-α by NK cells. CONCLUSIONS: This study highlights IL-26 as a new player in the inflammatory and antiviral immune responses associated with chronic HCV infection.

Dendritic cells and vaccine design for sexually-transmitted diseases.

Dendritic cells (DCs) are major antigen presenting cells (APCs) that can initiate and control host immune responses toward either immunity or tolerance. These features of DCs, as immune orchestrators, are well characterized by their tissue localizations as well as by their subset-dependent functional specialties and plasticity. Thus, the level of protective immunity to invading microbial pathogens can be dependent on the subsets of DCs taking up microbial antigens and their functional plasticity in response to microbial products, host cellular components and the cytokine milieu in the microenvironment. Vaccines are the most efficient and cost-effective preventive medicine against infectious diseases. However, major challenges still remain for the diseases caused by sexually-transmitted pathogens, including HIV, HPV, HSV and Chlamydia. We surmise that the establishment of protective immunity in the female genital mucosa, the major entry and transfer site of these pathogens, will bring significant benefit for the protection against sexually-transmitted diseases. Recent progresses made in DC biology suggest that vaccines designed to target proper DC subsets may permit us to establish protective immunity in the female genital mucosa against sexually-transmitted pathogens.

Origin, Phenotype, and Function of Mouse Dendritic Cell Subsets.

Dendritic cells are cells of hematopoietic origin that are specialized in antigen presentation and instruction of innate and adaptive immune responses. They are a heterogenous group of cells populating lymphoid organs and most tissues. Dendritic cells are commonly separated in three main subsets that differ in their developmental paths, phenotype, and functions. Most studies on dendritic cells were done primarily in mice; therefore, in this chapter, we propose to summarize the current knowledge and recent progress on mouse dendritic cell subsets' development, phenotype, and functions.

Enrichment of Large Numbers of Splenic Mouse Dendritic Cells After Injection of Flt3L-Producing Tumor Cells.

Dendritic cells (DCs) are antigen-presenting cells (APCs) that shape innate and adaptive immunity. There are multiple subsets of DCs distinguished according to their phenotype and functional specialization. DCs are present in lymphoid organs and across multiple tissues. However, their frequency and numbers at these sites are very low making their functional study difficult. Multiple protocols have been developed to generate DCs in vitro from bone marrow progenitors, but they do not fully recapitulate DC complexity found in vivo. Therefore, directly amplifying endogenous DCs in vivo appears as an option to overcome this specific caveat. In this chapter, we describe a protocol to amplify murine DCs in vivo by the injection of a B16 melanoma cell line expressing the trophic factor FMS-like tyrosine kinase 3 ligand (Flt3L). We have also compared two methods of magnetic sorting of amplified DCs, both giving high yields of total murine DCs, but different representation of the main DC subsets found in vivo.

Elevated levels of circulatory follicular T helper cells in chronic lymphocytic leukemia contribute to B cell expansion.

Chronic lymphocytic leukemia (CLL) is characterized by an expansion of mature B cells in the bone marrow, peripheral lymphoid organs, and blood. CD4 T helper (Th) lymphocytes significantly contribute to the physiopathology of CLL, but the subset(s) of Th cell involved in CLL pathogenesis is (are) still under debate. In this study, we performed flow cytometry analysis of the circulatory T cells of untreated CLL patients and observed an increase in follicular helper T cells (Tfh), which is a subset of T cells specialized in B cell help. Elevated numbers of Tfh cells correlated with disease severity as measured by the Binet staging system. Tfh from CLL patients were activated and skewed toward a Th1 profile as evidenced by their PD-1+IL-21+IFNγ+ phenotype and their CXCR3+CCR6- chemokine receptor profile. Tfh efficiently enhanced B-CLL survival and proliferation through IL-21 but independently of IFNγ. Finally, we observed an inverse correlation between the Tfh1 and IgA and IgG serum levels in patients, suggesting a role for this Tfh subset in the immune dysfunction associated with CLL. Altogether, our data highlight an impairment in circulatory Tfh subsets in CLL patients and their critical role in CLL physiopathology.

Concomitant activation and antigen uptake via human dectin-1 results in potent antigen-specific CD8+ T cell responses.

Dectin-1, a C-type lectin recognizing fungal and mycobacterial pathogens, can deliver intracellular signals that activate dendritic cells (DCs), resulting in initiation of immune responses and expansion of Th17 CD4(+) T cell responses. In this paper, we studied the roles of human Dectin-1 (hDectin-1) expressed on DCs in the induction and activation of Ag-specific CD8(+) T cell responses. We first generated an agonistic anti-hDectin-1 mAb, which recognizes the hDectin-1 Glu(143)-Ile(162) region. It bound to in vitro monocyte-derived DCs and to in vivo CD1c(+)CD1a(+) dermal DCs but not to epidermal Langerhans cells. Anti-hDectin-1-mediated DC activation resulted in upregulation of costimulatory molecules and secretion of multiple cytokines and chemokines in a Syk-dependent manner. DCs activated with the anti-hDectin-1 mAb could significantly enhance both neo and foreign Ag-specific CD8(+) T cell responses by promoting both the expansion of CD8(+) T cells and their functional activities. We further demonstrated that delivering Ags to DCs via hDectin-1 using anti-hDectin-1-Ag conjugates resulted in potent Ag-specific CD8(+) T cell responses. Thus, hDectin-1 expressed on DCs can contribute to the induction and activation of cellular immunity against intracellular pathogens, such as mycobacteria, that are recognized by DCs via Dectin-1. Vaccines based on delivering Ags to DCs with an agonistic anti-hDectin-1 mAb could elicit CD8(+) T cell-mediated immunity.

Differential expression of nuclear hormone receptors by dendritic cell subsets in human vaginal mucosa and skin.

Nuclear hormone receptors (NHRs) expressed by dendritic cells (DCs), the major immune inducers and regulators, could play important roles in host immunity. Assessment of NHRs expressed by DCs in the vaginal mucosa (VM), in comparison with those expressed by DCs in other tissues, will thus help us understand the immunology of human vagina. This study identified 16 NHR transcripts that are differentially expressed among 8 different antigen-presenting cell (APC) subsets isolated from human VM, skin, and blood. The expression profiles of NHRs were largely tissue specific. VM APCs expressed increased levels of LXRA, RXRA, ESRRA, ESRRAP2, and PPARG, whereas skin and blood APCs expressed increased levels of NURR1, NOR1 and RARA. Of interest, female sex hormone receptors, ESR1 and PGR, were found to be mainly expressed by non-APC cell types in the VM; ESR1 by HLA-DR+CD34+ and PGR by HLA-DR- cells. ERα and PR were expressed by vimentin+ cells in the VM, but not in human skin. ERα, but not PR, was also expressed in CD10+ cells in the lamina propria of VM. In conclusion, NHR expression by APC subsets is tissue- and cell type-specific. Future studies on the roles of individual NHRs expressed by different cell types, including DC subsets, in the human VM are warranted.

Interleukin-1ß-Activated Microvascular Endothelial Cells Promote DC-SIGN-Positive Alternatively Activated Macrophages as a Mechanism of Skin Fibrosis in Systemic Sclerosis.

OBJECTIVE: To characterize the role of interleukin-1ß (IL-1ß) and microvascular endothelial cells (MVECs) in the generation of alternatively activated macrophages in the skin, and to explore their role in the development of skin fibrosis in patients with systemic sclerosis (SSc; scleroderma). METHODS: Conditioned medium prepared with MVECs purified from the skin of healthy donors and the skin of SSc patients was used to generate monocyte-derived macrophages. Flow cytometry, multiplex protein assessment, real-time quantitative polymerase chain reaction, and tissue immunofluorescence were used to characterize MVEC-induced polarization of alternatively activated macrophages. Coculture experiments were conducted to assess the role of MVEC-induced alternatively activated macrophages in fibroblast activation. Alternatively activated macrophages were characterized in the skin of healthy donors and SSc patients using multiparametric immunofluorescence and multiplex immunostaining for gene expression. Based on our in vitro data, we defined a supervised macrophage gene signature score to assess correlation between the macrophage score and clinical features in patients with SSc, using the Spearman's test. RESULTS: IL-1ß-activated MVECs from SSc patients induced monocytes to differentiate into DC-SIGN+ alternatively activated macrophages producing high levels of CCL18, CCL2, and CXCL8 but low levels of IL-10. DC-SIGN+ alternatively activated macrophages showed significant enhancing effects in promoting the production of proinflammatory fibroblasts and were found to be enriched in perivascular regions of the skin of SSc patients who had a high fibrosis severity score. A novel skin transcriptomic macrophage signature, defined from our in vitro findings, correlated with the extent of skin fibrosis (Spearman's r = 0.6, P = 0.0018) and was associated with early disease manifestations and lung involvement in patients with SSc. CONCLUSION: Our findings shed new light on the vicious circle implicating unabated IL-1ß secretion, MVEC activation, and the generation of DC-SIGN+ alternatively activated macrophages in the development of skin fibrosis in patients with SSc.

The IL-27 p28 subunit binds cytokine-like factor 1 to form a cytokine regulating NK and T cell activities requiring IL-6R for signaling.

IL-27 is formed by the association of a cytokine subunit, p28, with the soluble cytokine receptor EBV-induced gene 3 (EBI3). The IL-27R comprises gp130 and WSX-1. The marked difference between EBI3(-/-) and WSX-1(-/-) mice suggests that p28 has functions independent of EBI3. We have identified an alternative secreted complex formed by p28 and the soluble cytokine receptor cytokine-like factor 1 (CLF). Like IL-27, p28/CLF is produced by dendritic cells and is biologically active on human NK cells, increasing IL-12- and IL-2-induced IFN-gamma production and activation marker expression. Experiments with Ba/F3 transfectants indicate that p28/CLF activates cells expressing IL-6Ralpha in addition to the IL-27R subunits. When tested on CD4 and CD8 T cells, p28/CLF induces IL-6Ralpha-dependent STAT1 and STAT3 phosphorylation. Furthermore, p28/CLF inhibits CD4 T cell proliferation and induces IL-17 and IL-10 secretion. These results indicate that p28/CLF may participate in the regulation of NK and T cell functions by dendritic cells. The p28/CLF complex engages IL-6R and may therefore be useful for therapeutic applications targeting cells expressing this receptor. Blocking IL-6R using humanized mAbs such as tocilizumab has been shown to be beneficial in pathologies like rheumatoid arthritis and juvenile idiopathic arthritis. The identification of a new IL-6R ligand is therefore important for a complete understanding of the mechanism of action of this emerging class of immunosuppressors.

PolyI:C plus IL-2 or IL-12 induce IFN-gamma production by human NK cells via autocrine IFN-beta.

NK lymphocytes and type I IFN (IFN-alpha/beta) are major actors of the innate anti-viral response that also influence adaptive immune responses. We evaluated type I IFN production by human NK cells in response to polyI:C, a potent type I IFN-inducing TLR3 agonist. PolyI:C plus IL-2/IL-12 induced IFN-beta (but not IFN-alpha) mRNA expression and protein production by highly pure human NK cells and by the human NK cell line NK92. Neutralizing anti-IFNAR1 or anti-IFN-beta Ab prevented the production of IFN-gamma induced by polyI:C plus IL-2/IL-12. Similarly, IFN-gamma production induced by polyI:C plus IL-12 was reduced in NK cells isolated from IFNAR1(-/-) compared with WT mice. The ability of polyI:C plus IL-12 to induce IFN-gamma production was related to an increase of TLR3, Mda5 and IFNAR expression and by an increase of STAT1 and STAT4 phosphorylation. Collectively, these data demonstrate that NK cells, in response to polyI:C plus IL-2/IL-12, produce IFN-beta that induce, in an autocrine manner, the production of IFN-gamma and thereby highlight that NK cells may control the outcome of protective or injurious immune responses through type I IFN secretion.

Functions of Tfh Cells in Common Variable Immunodeficiency.

Common variable immunodeficiency is the most common clinical primary immunodeficiency in adults. Its hallmarks are hypogammaglobulinemia and compromised B-cell differentiation into memory or antibody-secreting cells leading to recurrent infections. This disease is heterogeneous, with some patients harboring multiple complications such as lymphoproliferative disorders, autoimmune manifestations, or granulomatous inflammation. The mechanisms leading to these complications remain elusive despite numerous associations found in the literature. For instance, although described as a B cell intrinsic disease, numerous abnormalities have been reported in other immune cell compartments. Here, we tuned our attention to follicular helper T cells, a CD4+ T cell population specialized in B cell help, considering the recent publications showing an involvement of these cells in CVID pathogenesis.

Interferon-gamma reverses the immunosuppressive and protumoral properties and prevents the generation of human tumor-associated macrophages.

Tumor-associated macrophages (TAM) are M2d-polarized cells (IL-10(high), IL-12(low), ILT3(high), CD86(low)) that accumulate in tumor microenvironment. TAM inhibit antitumor T lymphocyte generation and function, contribute to tumor tolerance and are trophic for tumors. In this study, we investigated whether some immunological factors may reverse TAM immunosuppressive properties. Among 32 cytokines, we have identified IFNgamma on its ability to switch immunosuppressive TAM into immunostimulatory cells. Upon IFNgamma exposure, TAM purified from ovarian cancer ascites recover a M1 phenotype (IL-10(low), IL-12(high)), express high levels of CD86 and low levels of ILT3, enhance the proliferation of CD4(+) T lymphocytes and potentiate the cytotoxic properties of a MelanA-specific CD8(+) T cell clone. IFNgamma-treated TAM also secreted reduced levels of mediators promoting suppressive T cell accumulation (CCL18) and trophic for tumors (VEGF and MMP9). As TAM derive from the local differentiation of peripheral blood monocytes, we investigated whether IFNgamma may also affect TAM generation. In the presence of ovarian ascites, IFNgamma skewed monocyte differentiation from TAM-like cells to M1-polarized immunostimulatory macrophages. Together, these data show that IFNgamma overcomes TAM-induced immunosuppression by preventing TAM generation and functions. These data highlight that IFNgamma used locally at the tumor site could potentiate the efficacy of antitumor immunotherapies based on the generation of effector T cells.

The Multiple Layers of the Tumor Environment.

The notion of tumor microenvironment (TME) has been brought to the forefront of recent scientific literature on cancer. However, there is no consensus on how to define and spatially delineate the TME. We propose that the time is ripe to go beyond an all-encompassing list of the components of the TME, and to construct a multilayered view of cancer. We distinguish six layers of environmental interactions with the tumor and show that they are associated with distinct mechanisms, and ultimately with distinct therapeutic approaches.