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Background: Neoantigen reactive T cell (NRT) has the ability to inhibit the growth of tumors expressing specific neoantigens. However, due to the difficult immune infiltration and the inhibition of tumor microenvironment, the therapeutic effect of NRT in solid tumors is limited. In this study, we designed NRT cells (7×19 NRT) that can express both interleukin-7 (IL-7) and chemokine C-C motif ligand 19 (CCL19) in mouse lung cancer cells, and evaluated the difference in anti-tumor effect between 7×19 NRT cells and conventional NRT cells., Methods: We performed next-generation sequencing and neoantigen prediction for mouse Lewis lung carcinoma (LLC), prepared RNA vaccine, cultured NRT cells, constructed retroviral vectors encoding IL-7 and CCL19, transduced NRT cells and IL-7 and CCL19 were successfully expressed, and 7×19 NRT was successfully obtained. The anti-tumor effect was evaluated in vivo and in vitro in mice., Results: The 7×19 NRT cells significantly enhanced the proliferation and invasion ability of T cells by secreting IL-7 and CCL19, achieved significant tumor inhibition in the mouse lung cancer and extended the survival period of mice. The T cell infiltration into tumor tissue and the necrosis of tumor tissue increased significantly after 7×19 NRT treatment. In addition, both 7×19 NRT treatment and conventional NRT treatment were safe., Conclusions: The anti-solid tumor ability of NRT cells is significantly enhanced by the arming of IL-7 and CCL19, which is a safe and effective genetic modification of NRT.

CC chemokine ligand 19 (CCL19) plays a key role in the regulation of immune responses including homeostasis, inflammation, and immune tolerance. In this study, two variants of CCL19 homologues (CCL19a2 and CCL19b) and CCR7 were investigated in grass carp Ctenopharyngodon idella. The three genes were widely expressed in immune tissues and could be modulated by stimulation with LPS, PHA and poly(I:C), and infection with Flavobacterium columnare and grass carp reovirus. In an in vitro chemotaxis assay, the recombinant CCL19a2 and CCL19b were active to promote the migration of HEK293 T cells expressing CCR7 and leucocytes isolated from the gills, head kidney and spleen. Moreover, their chemotactive effects were validated in vivo. We found that the cells recruited by CCL19a2 and CCl19b are mainly monocytes/macrophages expressing high levels of IL-1β, IFN-γ, colony stimulating factor 1 receptor (CSF1R) and MHC II. Our work suggests that CCL19a2 and CCl19b are involved in recruitment of antigen presenting cells in fish., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Potent systemic immunity is important for recalled mucosal immune responses, but in the defense against mucosal viral infections, it usually remains low at mucosal sites. Based on our previous findings that enhanced immune responses can be achieved by immunization with an immunogen in combination with a molecular adjuvant, here we designed chemokine-antigen (Ag) fusion constructs (CCL19- or CCL28-herpes simplex virus 2 glycoprotein D [HSV-2 gD]). After intramuscular (i.m.) immunization with different DNA vaccines in a prime and boost strategy, BALB/c mice were challenged with a lethal dose of HSV-2 through the genital tract. Ag-specific immune responses and chemokine receptor-specific lymphocytes were analyzed to determine the effects of CCL19 and CCL28 in strengthening humoral and cellular immunity. Both CCL19 and CCL28 were efficient in inducing long-lasting HSV-2 gD-specific systemic immunity. Compared to CCL19, less CCL28 was required to elicit HSV-2 gD-specific serum IgA responses, Th1- and Th2-like responses of immunoglobulin (Ig) subclasses and cytokines, and CCR3 + T cell enrichment (>8.5-fold) in spleens. These findings together demonstrate that CCL28 tends to assist an immunogen to induce more potently protective immunity than CCL19. This work provides information for the application potential of a promising vaccination strategy against mucosal infections caused by HSV-2 and other sexually transmitted viruses. IMPORTANCE An effective HSV-2 vaccine should induce antigen (Ag)-specific immune responses against viral mucosal infection. This study reveals that chemokine CCL19 or CCL28 enhanced HSV-2 glycoprotein D ectodomain (gD-306aa)-induced immune responses against vaginal virus challenge. In addition to eliciting robust humoral immune responses, the chemokine-Ag fusion construct also induced Th1- and Th2-like immune responses characterized by the secretion of multiple Ig subclasses and cytokines that were able to be recalled after HSV-2 challenge, while CCL28 appeared to be more effective than CCL19 in promoting gD-elicited immune responses as well as the migration of T cells to secondary lymph tissues. Of importance, both CCL19 and CCL28 significantly facilitated gD to induce protective mucosal immune responses in the genital tract. The above-described findings together highlight the potential of CCL19 or CCL28 in combination with gD as a vaccination strategy to control HSV-2 infection., (Copyright © 2021 Yan et al.)

Objective: Juvenile dermatomyositis (DM) is a heterogeneous systemic immune-mediated vasculopathy. This study was undertaken to 1) identify inflammation/endothelial dysfunction-related biomarker profiles reflecting disease severity at diagnosis, and 2) establish whether such biomarker profiles could be used for predicting the response to treatment in patients with juvenile DM., Methods: In total, 39 biomarkers related to activation of endothelial cells, endothelial dysfunction, and inflammation were measured using multiplex technology in serum samples from treatment-naive patients with juvenile DM from 2 independent cohorts (n = 30 and n = 29). Data were analyzed by unsupervised hierarchical clustering, nonparametric tests with correction for multiple comparisons, and Kaplan-Meier tests with Cox proportional hazards models for analysis of treatment duration. Myositis-specific antibodies (MSAs) were measured in the patients' serum using line blot assays., Results: Severe vasculopathy in patients with juvenile DM was associated with low serum levels of intercellular adhesion molecule 1 (Spearman's rho [r s ] = 0.465, P = 0.0111) and high serum levels of endoglin (r s = -0.67, P < 0.0001). In the discovery cohort, unsupervised hierarchical clustering analysis of the biomarker profiles yielded 2 distinct patient clusters, of which the smaller cluster (cluster 1; n = 8) exhibited high serum levels of CXCL13, CCL19, galectin-9, CXCL10, tumor necrosis factor receptor type II (TNFRII), and galectin-1 (false discovery rate <0.0001), and this cluster had greater severity of muscle disease and global disease activity (each P < 0.05 versus cluster 2). In the validation cohort, correlations between the serum levels of galectin-9, CXCL10, TNFRII, and galectin-1 and the severity of global disease activity were confirmed (r s = 0.40-0.52, P < 0.05). Stratification of patients according to the 4 confirmed biomarkers identified a cluster of patients with severe symptoms (comprising 64.7% of patients) who were considered at high risk of requiring more intensive treatment in the first 3 months after diagnosis (P = 0.0437 versus other cluster). Moreover, high serum levels of galectin-9, CXCL10, and TNFRII were predictive of a longer total treatment duration (P < 0.05). The biomarker-based clusters were not evidently correlated with patients' MSA serotypes., Conclusion: Results of this study confirm the heterogeneity of new-onset juvenile DM based on serum biomarker profiles. Patients with high serum levels of galectin-9, CXCL10, TNFRII, and galectin-1 may respond suboptimally to conventional treatment, and may therefore benefit from more intensive monitoring and/or treatment., (© 2020 The Authors. Arthritis & Rheumatology published by Wiley Periodicals, Inc. on behalf of American College of Rheumatology.)

Despite significant therapeutic improvements chronic lymphocytic leukemia (CLL) remains an incurable disease and there is a persistent pursuit of new treatment alternatives. Lurbinectedin, a selective inhibitor of active transcription of protein-coding genes, is currently in phase II/III clinical trials for solid tumors such as small-cell lung cancer (SCLC). In this study, we aimed to evaluate the activity of Lurbinectedin on circulating mononuclear cells from CLL patients and to determine whether Lurbinectedin could affect the cross-talk between B-CLL cells and the tumor microenvironment. We found that Lurbinectedin induced a dose- and time-dependent death in all cell types evaluated, with B cells, monocytes and monocytic myeloid derived suppressor cells (Mo-MDSC) being the most susceptible populations. At sub-apoptotic doses, Lurbinectedin decreased the expression of CCR7 in B-CLL cells and impaired their migration towards CCL19 and CCL21. Furthermore, low concentrations of Lurbinectedin stimulated the synthesis of pro-IL1β in monocytes and nurse-like cells, without inducing the inflammasome activation. Altogether, these results indicate that Lurbinectedin might have antitumor activity in CLL due to its direct action on leukemic cells in combination with its effects on the tumor microenvironment. Our findings encourage further investigation of Lurbinectedin as a potential therapy for CLL.

Background: The chemokine MIP-3α (CCL20) binds to CCR6 on immature dendritic cells. DNA vaccines fusing MIP-3α to melanoma-associated antigens have shown improved efficacy and immunogenicity in the B16F10 mouse melanoma model. Here, we report that the combination of type-I interferon therapy (IFNα) with 5-Aza-2'-deoxycitidine (5Aza) profoundly enhanced the therapeutic efficacy of a MIP-3α-Gp100-Trp2 DNA vaccine., Methods: Beginning on day 5 post-transplantation of B16F10 melanoma, vaccine was administered intramuscularly (i.m.) by electroporation. CpG adjuvant was given 2 days later. 5Aza was given intraperitoneally at 1 mg/kg and IFNα therapy either intratumorally or i.m. as noted. Tumor sizes, tumor growth, and mouse survival were assessed. Tumor lysate gene expression levels and tumor-infiltrating lymphocytes (TILs) were assessed by qRT-PCR and flow cytometry, respectively., Results: Adding IFNα and 5Aza treatments to mice vaccinated with MIP-3α-Gp100-Trp2 leads to reduced tumor burden and increased median survival (39% over vaccine and 95% over controls). Tumor lysate expression of CCL19 and CCR7 were upregulated ten and fivefold over vaccine, respectively. Vaccine-specific and overall CD8+ TILs were increased over vaccine (sevenfold and fourfold, respectively), as well as the proportion of TILs that were CD8+ (twofold)., Conclusions: Efficient targeting of antigen to immature dendritic cells with a chemokine-fusion vaccine offers an alternative to classic and dendritic cell vaccines. Combining this approach with IFNα and 5Aza treatment significantly improved vaccine efficacy. This improved efficacy correlated with changes in chemokine gene expression and CD8+ TIL infiltration and was dependent on the presence of all therapeutic components.

Lymph nodes (LNs) are at the cross roads of immunity and tolerance. These tissues are compartmentalized into specialized niche areas by lymph node stromal cells (LN SCs). LN SCs shape the LN microenvironment and guide immunological cells into different zones through establishment of a CCL19 and CCL21 gradient. Following local immunological cues, LN SCs modulate activity to support immune cell priming, activation, and fate. This review will present our current understanding of LN SC subsets roles in regulating T cell tolerance. Three major types of LN SC subsets, namely fibroblastic reticular cells, lymphatic endothelial cells, and blood endothelial cells, are discussed. These subsets serve as scaffolds to support and regulate T cell homeostasis. They contribute to tolerance by presenting peripheral tissue antigens to both CD4 and CD8 T cells. The role of LN SCs in regulating T cell migration and tolerance induction is discussed. Looking forward, recent advances in bioengineered materials and approaches to leverage LN SCs to induce T cell tolerance are highlighted, as are current clinical practices that allow for manipulation of the LN microenvironment to induce tolerance. Increased understanding of LN architecture, how different LN SCs integrate immunological cues and shape immune responses, and approaches to induce T cell tolerance will help further combat autoimmune diseases and graft rejection., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

All protective and pathogenic immune and inflammatory responses rely heavily on leukocyte migration and localization. Chemokines are secreted chemoattractants that orchestrate the positioning and migration of leukocytes through concentration gradients. The mechanisms underlying chemokine gradient establishment and control include physical as well as biological phenomena. Mathematical models offer the potential to both understand this complexity and suggest interventions to modulate immune function. Constructing models that have powerful predictive capability relies on experimental data to estimate model parameters accurately, but even with a reductionist approach most experiments include multiple cell types, competing interdependent processes and considerable uncertainty. Therefore, we propose the use of reduced modeling and experimental frameworks in complement, to minimize the number of parameters to be estimated. We present a Bayesian optimization framework that accounts for advection and diffusion of a chemokine surrogate and the chemokine CCL19, transport processes that are known to contribute to the establishment of spatio-temporal chemokine gradients. Three examples are provided that demonstrate the estimation of the governing parameters as well as the underlying uncertainty. This study demonstrates how a synergistic approach between experimental and computational modeling benefits from the Bayesian approach to provide a robust analysis of chemokine transport. It provides a building block for a larger research effort to gain holistic insight and generate novel and testable hypotheses in chemokine biology and leukocyte trafficking., (Copyright © 2019 Kalogiros, Russell, Bonneuil, Frattolin, Watson, Moore, Kypraios and Brook.)

Chemokine receptors play important roles in the immune system and are linked to several human diseases. Targeting chemokine receptors have so far shown very little success owing to, to some extent, the promiscuity of the immune system and the high degree of biased signaling within it. CCR7 and its two endogenous ligands display biased signaling and here we investigate the differences between the two ligands, CCL21 and CCL19, with respect to their biased activation of CCR7. We use bystander bioluminescence resonance energy transfer (BRET) based signaling assays and Transwell migration assays to determine (A) how swapping of domains between the two ligands affect their signaling patterns and (B) how receptor mutagenesis impacts signaling. Using chimeric ligands we find that the chemokine core domains are central for determining signaling outcome as the lack of β-arrestin-2 recruitment displayed by CCL21 is linked to its core domain and not N-terminus. Through a mutagenesis screen, we identify the extracellular domains of CCR7 to be important for both ligands and show that the two chemokines interact differentially with extracellular loop 2 (ECL-2). By using in silico modeling, we propose a link between ECL-2 interaction and CCR7 signal transduction. Our mutagenesis study also suggests a lysine in the top of TM3, K130 3.26 , to be important for G protein signaling, but not β-arrestin-2 recruitment. Taken together, the bias in CCR7 between CCL19 and CCL21 relies on the chemokine core domains, where interactions with ECL-2 seem particularly important. Moreover, TM3 selectively regulates G protein signaling as found for other chemokine receptors., (Copyright © 2019 Jørgensen, Larsen, Uetz-von Allmen, Lückmann, Legler, Frimurer, Veldkamp, Hjortø and Rosenkilde.)

The spleen is an important secondary lymph organ. Splenomegaly induced by anemia could affect the function of spleen in immune responses. We observe that anemia induced in mice with reduced peripheral T cell trafficking to the spleen T cell zones as well as CCL21 and CCL19 expression. In accordance with previous research, we found that the production of EPO in the mice kidney was sharply increased post anemia. In addition, mice were injected with different doses of EPO. Our results show that with the increased dosage of EPO, the chemokine expression in the spleen is lowered with a decrease in peripheral T cell homing to the spleen T cell zones. At last, our results show that the anemia mice model administrated with anti-EPO antibody had a higher expression of spleen CCL19 and CCL21 and an increased count of periphery T cells trafficking to spleen T cell zones at day 3 post induction. These data indicate that anemia could disturb T cell movement in the spleen, which might further affect T cell immune response, with partial involvement of EPO., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Immune checkpoint blockade therapy has changed prognoses for many melanoma patients. However, immune responses that correlate with clinical progression of the disease are still poorly understood. To identify immune responses correlating with melanoma clinical evolution, we analyzed serum cytokines as well as circulating NK and T-cell subpopulations from melanoma patients. The patients' immune profiles suggested that melanoma progression leads to changes in peripheral blood NK and T-cell subsets. Stage IV melanoma was characterized by an increased frequency of CCR7 + CD56 bright NK cells as well as high serum concentrations of the CCR7 ligand CCL19. CCR7 expression and CCL19 secretion were also observed in melanoma cell lines. The CCR7 + melanoma cell subpopulation coexpressed PD-L1 and Galectin-9 and had stemness properties. Analysis of melanoma-derived cancer stem cells (CSC) showed high CCR7 expression; these CSCs were efficiently recognized and killed by NK cells. An accumulation of CCR7 + , PD-L1 + , and Galectin-9 + melanoma cells in melanoma metastases was demonstrated ex vivo Altogether, our data identify biomarkers that may mark a CCR7-driven metastatic melanoma pathway., (©2019 American Association for Cancer Research.)

The infection by dengue virus (DENV) of microglia causes cell activation and migration via a mechanism involving viral entry, RNA release, and Toll-like receptor 3 signaling. In this study, we demonstrated that secreted chemotactic factors present in microglial conditioned medium (MCM) facilitated cell motility in the murine BV2 microglial cells. The pharmacological disruption of lipid rafts/caveolae reduced DENV- and ultraviolet (UV)-inactivated MCM-induced microglial cell migration. An antibody-based cytokine/chemokine array showed an increase in macrophage inflammatory protein (MIP)-3β in MCM produced using DENV-infected cells. The pharmacological inhibition of c-Jun N-terminal kinase (JNK) retarded UV-MCM-induced microglial cell migration. These results demonstrate that secreted MIP-3β and its effect on the JNK signaling pathways mediates DENV-induced BV2 microglial cell migration.

Background: A particular characteristic of non-small cell lung cancer is the composition of the tumor microenvironment with a very high proportion of fibroblastic stromal cells (FSCs)., Objective: Lapses in our basic knowledge of fibroblast phenotype and function in the tumor microenvironment make it difficult to define whether FSC subsets exist that exhibit either tumor-promoting or tumor-suppressive properties., Methods: We used gene expression profiling of lung versus tumor FSCs from patients with non-small cell lung cancer. Moreover, CCL19-expressing FSCs were studied in transgenic mouse models by using a lung cancer metastasis model., Results: CCL19 mRNA expression in human tumor FSCs correlates with immune cell infiltration and intratumoral accumulation of CD8 + T cells. Mechanistic dissection in murine lung carcinoma models revealed that CCL19-expressing FSCs form perivascular niches to promote accumulation of CD8 + T cells in the tumor. Targeted ablation of CCL19-expressing tumor FSCs reduced immune cell recruitment and resulted in unleashed tumor growth., Conclusion: These data suggest that a distinct population of CCL19-producing FSCs fosters the development of an immune-stimulating intratumoral niche for immune cells to control cancer growth., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Stromal cells (SCs) establish the compartmentalization of lymphoid tissues critical to the immune response. However, the full diversity of lymph node (LN) SCs remains undefined. Using droplet-based single-cell RNA sequencing, we identified nine peripheral LN non-endothelial SC clusters. Included are the established subsets, Ccl19 hi T-zone reticular cells (TRCs), marginal reticular cells, follicular dendritic cells (FDCs), and perivascular cells. We also identified Ccl19 lo TRCs, likely including cholesterol-25-hydroxylase + cells located at the T-zone perimeter, Cxcl9 + TRCs in the T-zone and interfollicular region, CD34 + SCs in the capsule and medullary vessel adventitia, indolethylamine N-methyltransferase + SCs in the medullary cords, and Nr4a1 + SCs in several niches. These data help define how transcriptionally distinct LN SCs support niche-restricted immune functions and provide evidence that many SCs are in an activated state., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The CC motif chemokine 19 (CCL19) functions in acute inflammation by recruiting lymphocytes and other cells. However, CCL19 has only been investigated in few fish species. In this study, we characterized a CCL19-like molecule (PaCCL19l) in ayu (Plecoglossus altivelis), a teleost fish. Sequence analysis revealed that PaCCL19l was most closely related to Atlantic salmon (Salmon salar) CCL19l1, which belonged to the fish CCL19a.1 subcluster. PaCCL19l was constitutively expressed in the tested ayu tissues and peripheral blood mononuclear cells (PBMCs), with the highest transcript level in PBMCs. Upon infection with Vibrio anguillarum, the expressions of PaCCL19l in the head kidney, liver, spleen, PBMCs, and monocytes/macrophages (MO/MΦ) were dramatically up-regulated. Recombinant PaCCL19l (rPaCCL19l) exhibited a significant effect on the chemotaxis of lymphocytes and MO/MΦ in vitro and in vivo. Meanwhile, rPaCCL19l exerted a high chemotaxic activity for lipopolysaccharide (LPS)-stimulated MO/MΦ (M1-type), but not for cyclic adenosine monophosphate (cAMP)-stimulated MO/MΦ (M2-type). When ayu MO/MΦ was treated with rPaCCL19l along with Vibrio anguillarum infection, the mRNA expression of proinflammatory cytokines (IL-1β, TNFα, IL-6, IL-12b, and IFN-γ) was up-regulated, while that of anti-inflammatory cytokines (IL-10, TGFβ, and IL-22) was down-regulated. Ayu MO/MΦ treated with anti-PaCCL19l IgG gave the opposite result. These results implicated that PaCCL19l is involved in the selective chemotaxis of ayu immune cells and promotes the host at a pro-inflammatory state., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The spleen is known as an important filter for blood-borne pathogens that are trapped by specialized macrophages in the marginal zone (MZ): the CD209 + MZ macrophages (MZMs) and the CD169 + marginal metallophilic macrophages (MMMs). Acute systemic infection strongly impacts MZ populations and the location of T and B lymphocytes. This phenomenon has been linked to reduced chemokine secretion by stromal cells. Brucella spp. are the causative agent of brucellosis, a widespread zoonotic disease. Here, we used Brucella melitensis infection as a model to investigate the impact of chronic stealth infection on splenic MZ macrophage populations. During the late phase of Brucella infection, we observed a loss of both MZMs and MMMs, with a durable disappearance of MZMs, leading to a reduction of the ability of the spleen to take up soluble antigens, beads, and unrelated bacteria. This effect appears to be selective as every other lymphoid and myeloid population analyzed increased during infection, which was also observed following Brucella abortus and Brucella suis infection. Comparison of wild-type and deficient mice suggested that MZ macrophage population loss is dependent on interferon gamma (IFN-γ) receptor but independent of T cells or tumor necrosis factor alpha receptor 1 (TNF-αR1) signaling pathways and is not correlated to an alteration of CCL19, CCL21, and CXCL13 chemokine mRNA expression. Our results suggest that MZ macrophage populations are particularly sensitive to persistent low-level IFN-γ-mediated inflammation and that Brucella infection could reduce the ability of the spleen to perform certain MZM- and MMM-dependent tasks, such as antigen delivery to lymphocytes and control of systemic infection., (Copyright © 2017 American Society for Microbiology.)

The chemokine receptor CCR7 drives leukocyte migration into and within lymph nodes (LNs). It is activated by chemokines CCL19 and CCL21, which are scavenged by the atypical chemokine receptor ACKR4. CCR7-dependent navigation is determined by the distribution of extracellular CCL19 and CCL21, which form concentration gradients at specific microanatomical locations. The mechanisms underpinning the establishment and regulation of these gradients are poorly understood. In this article, we have incorporated multiple biochemical processes describing the CCL19-CCL21-CCR7-ACKR4 network into our model of LN fluid flow to establish a computational model to investigate intranodal chemokine gradients. Importantly, the model recapitulates CCL21 gradients observed experimentally in B cell follicles and interfollicular regions, building confidence in its ability to accurately predict intranodal chemokine distribution. Parameter variation analysis indicates that the directionality of these gradients is robust, but their magnitude is sensitive to these key parameters: chemokine production, diffusivity, matrix binding site availability, and CCR7 abundance. The model indicates that lymph flow shapes intranodal CCL21 gradients, and that CCL19 is functionally important at the boundary between B cell follicles and the T cell area. It also predicts that ACKR4 in LNs prevents CCL19/CCL21 accumulation in efferent lymph, but does not control intranodal gradients. Instead, it attributes the disrupted interfollicular CCL21 gradients observed in Ackr4 -deficient LNs to ACKR4 loss upstream. Our novel approach has therefore generated new testable hypotheses and alternative interpretations of experimental data. Moreover, it acts as a framework to investigate gradients at other locations, including those that cannot be visualized experimentally or involve other chemokines., (Copyright © 2017 The Authors.)

Context: Increasing evidences suggest a correlation between gut and type 1 diabetes (T1D)., Objective: The objective of this study is to evaluate the gut inflammatory profile and microbiota in patients with T1D compared with healthy control (CTRL) subjects and patients with celiac disease (CD) as gut inflammatory disease controls., Design/setting/participants: The inflammatory status and microbiome composition were evaluated in biopsies of the duodenal mucosa of patients with T1D (n = 19), in patients with CD (n = 19), and CTRL subjects (n = 16) recruited at San Raffaele Scientific Institute, in Milan, Italy, between 2009 and 2015., Main Outcome Measures: Inflammation was evaluated by gene expression study and immunohistochemistry. Microbiome composition was analyzed by 16S ribosomal RNA gene sequencing., Results: An increased expression of CCL13, CCL19, CCL22, CCR2, COX2, IL4R, CD68, PTX3, TNFα, and VEGFA was observed in patients with T1D compared with CTRL subjects and patients with CD. Immunohistochemical analysis confirmed T1D-specific inflammatory status compared with healthy and CD control tissues, mainly characterized by the increase of the monocyte/macrophage lineage infiltration. The T1D duodenal mucosal microbiome results were different from the other groups, with an increase in Firmicutes and Firmicutes/Bacteroidetes ratio and a reduction in Proteobacteria and Bacteroidetes. The expression of genes specific for T1D inflammation was associated with the abundance of specific bacteria in the duodenum., Conclusions: This study shows that duodenal mucosa in T1D presents disease-specific abnormalities in the inflammatory profile and microbiota. Understanding the mechanisms underlying these features is critical to disentangle the complex pathogenesis of T1D and to gain new perspectives for future therapies targeting the intestine., (Copyright © 2017 by the Endocrine Society)

HIV vaginal transmission accounts for the majority of newly acquired heterosexual infections. However, the mechanism by which HIV spreads from the initial site of viral entry at the mucosal surface of the female genital tract to establish a systemic infection of lymphoid and peripheral tissues is not known. Once the virus exits the mucosa it rapidly spreads to all tissues, leading to CD4 + T cell depletion and the establishment of a viral reservoir that cannot be eliminated with current treatments. Understanding the molecular and cellular requirements for viral dissemination from the genital tract is therefore of great importance, as it could reveal new strategies to lengthen the window of opportunity to target the virus at its entry site in the mucosa where it is the most vulnerable and thus prevent systemic infection. Using HIV vaginal infection of humanized mice as a model of heterosexual transmission, we demonstrate that blocking the ability of leukocytes to respond to chemoattractants prevented HIV from leaving the female genital tract. Furthermore, blocking lymphocyte egress from lymph nodes prevented viremia and infection of the gut. Leukocyte trafficking therefore plays a major role in viral dissemination, and targeting the chemoattractant molecules involved can prevent the establishment of a systemic infection., Competing Interests: Conflict of interest: The authors have declared that no conflict of interest exists.

One major challenge in multiple sclerosis is to understand the cellular and molecular mechanisms leading to disease severity progression. The recently demonstrated correlation between disease severity and remyelination emphasizes the importance of identifying factors leading to a favourable outcome. Why remyelination fails or succeeds in multiple sclerosis patients remains largely unknown, mainly because remyelination has never been studied within a humanized pathological context that would recapitulate major events in plaque formation such as infiltration of inflammatory cells. Therefore, we developed a new paradigm by grafting healthy donor or multiple sclerosis patient lymphocytes in the demyelinated lesion of nude mice spinal cord. We show that lymphocytes play a major role in remyelination whose efficacy is significantly decreased in mice grafted with multiple sclerosis lymphocytes compared to those grafted with healthy donors lymphocytes. Mechanistically, we demonstrated in vitro that lymphocyte-derived mediators influenced differentiation of oligodendrocyte precursor cells through a crosstalk with microglial cells. Among mice grafted with lymphocytes from different patients, we observed diverse remyelination patterns reproducing for the first time the heterogeneity observed in multiple sclerosis patients. Comparing lymphocyte secretory profile from patients exhibiting high and low remyelination ability, we identified novel molecules involved in oligodendrocyte precursor cell differentiation and validated CCL19 as a target to improve remyelination. Specifically, exogenous CCL19 abolished oligodendrocyte precursor cell differentiation observed in patients with high remyelination pattern. Multiple sclerosis lymphocytes exhibit intrinsic capacities to coordinate myelin repair and further investigation on patients with high remyelination capacities will provide new pro-regenerative strategies., (© The Author (2017). Published by Oxford University Press on behalf of the Guarantors of Brain.)

Dendritic cells (DCs) play a pivotal role in the regulation of the immune response. DC development and activation is finely orchestrated through transcriptional programs. GATA1 transcription factor is required for murine DC development, and data suggest that it might be involved in the fine-tuning of the life span and function of activated DCs. We generated DC-specific Gata1 knockout mice (Gata1-KO DC ), which presented a 20% reduction of splenic DCs, partially explained by enhanced apoptosis. RNA sequencing analysis revealed a number of deregulated genes involved in cell survival, migration, and function. DC migration toward peripheral lymph nodes was impaired in Gata1-KO DC mice. Migration assays performed in vitro showed that this defect was selective for CCL21, but not CCL19. Interestingly, we show that Gata1-KO DC DCs have reduced polysialic acid levels on their surface, which is a known determinant for the proper migration of DCs toward CCL21., (Copyright © 2016 by The American Association of Immunologists, Inc.)

Mucosal surfaces require balancing different physiological roles and immune functions. To effectively achieve multifunctionality, mucosal epithelia have evolved unique microenvironments that create unique regional immune responses without impairing other normal physiological functions. Whereas examples of regional immunity are known in other mucosal epithelia, to date, no immune microenvironments have been described in the nasal mucosa, a site where the complex functions of olfaction and immunity need to be orchestrated. In this study we identified the presence of CD8α + cells in the rainbow trout (Oncorhynchus mykiss) nasal epithelium. Nasal CD8α + cells display a distinct phenotype suggestive of CD8 + T cells with high integrin β 2 expression. Importantly, nasal CD8α + cells are located in clusters at the mucosal tip of each olfactory lamella but scattered in the neuroepithelial region. The grouping of CD8α + cells may be explained by the greater expression of CCL19, ICAM-1, and VCAM-1 in the mucosal tip compared with the neuroepithelium. Whereas viral Ag uptake occurred via both tip and lateral routes, tip-resident MHC class II + cells are located significantly closer to the lumen of the nasal cavity than are their neuroepithelial counterparts, therefore having quicker access to invading pathogens. Our studies reveal compartmentalized mucosal immune responses within the nasal mucosa of a vertebrate species, a strategy that likely optimizes local immune responses while protecting olfactory sensory functions., Competing Interests: The authors have no financial conflicts of interest., (Copyright © 2016 by The American Association of Immunologists, Inc.)

Aim: CCL19 and its receptor CCR7 are essential molecules for facilitating the trafficking of mature dendritic cells (DCs) and helping to establish a microenvironment in lymphoid tissues to initiate primary immune responses, whereas CCL17 is required in the CCR7-CCL19-dependent migration of DCs. In this study we examined whether co-administration of CCL17 and CCL19 could enhance the immunogenicity of an anti-caries DNA vaccine, pCIA-P, in rodents., Methods: Plasmids encoding CCL17 (pCCL17/VAX) and CCL19 (pCCL19/VAX) were constructed. BALB/c mice were intranasally administered pCCL17/VAX, pCCL19/VAX, or pCCL17/VAX plus pCCL19/VAX, the migration of DCs to the spleen and draining lymph nodes (DLNs) was assessed with flow cytometry. The mice were co-administered pCIA-P; and the anti-PAc antibodies in the serum and saliva were detected with ELISA. Wistar rats were orally challenged with Streptococcus mutans and then administered pCIA-P in combination with pCCL17/VAX, pCCL19/VAX, or pCCL17/VAX plus pCCL19/VAX. The amount of S mutans sustained on rat molar surfaces was assessed using a colony forming assay. Caries activity was scored with the Keyes method., Results: Co-administration of the CCL17 and CCL19 genes in mice caused a greater increase in the number of mature DCs in the spleen and DLNs compared with administration of CCL17 or CCL19 genes alone. CCL17 and CCL19 double-adjuvant plus pCIA-P induced significantly higher levels of anti-PAc salivary IgA and anti-PAc serum IgG antibody in mice, and strengthened the ability of pCIA-P in inhibiting the colonization of S mutans on rat tooth surfaces. The caries activity of the combined adjuvant group was significantly lower than that of the pCCL17/VAX or the pCCL19/VAX group., Conclusion: A nasal adjuvant consisting of a combination of CCL17 and CCL19 attracts more mature DCs to secondary lymphoid tissues, inducing enhanced antibody responses against the anti-caries DNA vaccine pCIA-P and reducing S mutans infection in rodents.

We have previously demonstrated that prion protein-deficient (Prnp(0/0)) Zürich I mice display impaired T zone structure resulting from decreased splenic expression of the T cell homing chemokines, CCL19 and CCL21. Prions are transported to, and colonise in, the secondary lymphoid tissues. Therefore, in order to investigate how scrapie infection affects the splenic white pulp structure, we infected C57BL/6 mice with the mouse-adapted scrapie strain ME7 and analysed end-stage prion disease. We found that the white pulp regions of ME7-infected spleens were smaller, and contained markedly diminished T zones, as compared to control spleens. Although lymphoid tissue inducer cells were not affected, the expression of both CCL19 and CCL21 was decreased. In addition, the networks of follicular dendritic cells, which are known to express high levels of the cellular prion protein (PrP(C)) and to accumulate PrP(Sc) following scrapie infection, were larger in ME7-infected spleens. Further, they were associated with increased numbers of B cells expressing high levels of IgM. These data indicate that ME7-infected spleens display phenotype characteristics different from those reported for Prnp(0/0) spleens mainly due to the gain of PrP(Sc) function and suggest that the PrP(C) is required, not only to form the splenic white pulp structure, but also to maintain the intact T zone structure., (Copyright © 2016. Published by Elsevier GmbH.)

Fibroblastic reticular cells (FRCs) form the cellular scaffold of lymph nodes (LNs) and establish distinct microenvironmental niches to provide key molecules that drive innate and adaptive immune responses and control immune regulatory processes. Here, we have used a graph theory-based systems biology approach to determine topological properties and robustness of the LN FRC network in mice. We found that the FRC network exhibits an imprinted small-world topology that is fully regenerated within 4 wk after complete FRC ablation. Moreover, in silico perturbation analysis and in vivo validation revealed that LNs can tolerate a loss of approximately 50% of their FRCs without substantial impairment of immune cell recruitment, intranodal T cell migration, and dendritic cell-mediated activation of antiviral CD8+ T cells. Overall, our study reveals the high topological robustness of the FRC network and the critical role of the network integrity for the activation of adaptive immune responses.

Developing robust in vitro models of HIV latency is needed to better understand how latency is established, maintained and reversed. In this study, we examined the effects of donor variability, HIV titre and co-receptor usage on establishing HIV latency in vitro using two models of HIV latency. Using the CCL19 model of HIV latency, we found that in up to 50% of donors, CCL19 enhanced latent infection of resting CD4+ T-cells by CXCR4-tropic HIV in the presence of low dose IL-2. Increasing the infectious titre of CXCR4-tropic HIV increased both productive and latent infection of resting CD4+ T-cells. In a different model where myeloid dendritic cells (mDC) were co-cultured with resting CD4+ T-cells, we observed a higher frequency of latently infected cells in vitro than CCL19-treated or unstimulated CD4+ T-cells in the presence of low dose IL-2. In the DC-T-cell model, latency was established with both CCR5- and CXCR4-tropic virus but higher titres of CCR5-tropic virus was required in most donors. The establishment of latency in vitro through direct infection of resting CD4+ T-cells is significantly enhanced by CCL19 and mDC, but the efficiency is dependent on virus titre, co-receptor usage and there is significant donor variability.

The cellular prion protein is expressed in almost all tissues, including the central nervous system and lymphoid tissues. To investigate the effects of the prion protein in lymphoid cells and spleen structure formation, we used prion protein-deficient (Prnp(0/0)) Zürich I mice generated by inactivation of the Prnp gene. Prnp(0/0) mice had decreased lymphocytes, in particular, CD4 T cells and lymphoid tissue inducer (LTi) cells. Decreased CD4 T cells resulted from impaired expression of CCL19 and CCL21 in the spleen rather than altered chemokine receptor CCR7 expression. Importantly, some of the white pulp regions in spleens from Prnp(0/0) mice displayed impaired T zone structure as a result of decreased LTi cell numbers and altered expression of the lymphoid tissue-organizing genes lymphotoxin-α and CXCR5, although expression of the lymphatic marker podoplanin and CXCL13 by stromal cells was not affected. In addition, CD3(-)CD4(+)IL-7Rα(+) LTi cells were rarely detected in impaired white pulp in spleens of these mice. These data suggest that the prion protein is required to form the splenic white pulp structure and for development of normal levels of CD4 T and LTi cells., (Copyright © 2015. Published by Elsevier GmbH.)

There remain significant obstacles in developing biologics to treat primary biliary cholangitis (PBC). Although a number of agents have been studied both in murine models and human patients, the results have been relatively disappointing. IL-22 is a member of the IL-10 family and has multiple theoretical reasons for predicting successful usage in PBC. We have taken advantage of an IL-22 expressing adeno-associated virus (AAV-IL-22) to address the potential role of IL-22 in not only protecting mice from autoimmune cholangitis, but also in treating animals with established portal inflammation. Using our established mouse model of 2-OA-OVA immunization, including α-galactosylceramide (α-GalCer) stimulation, we treated mice both before and after the onset of clinical disease with AAV-IL-22. Firstly, AAV-IL-22 treatment given prior to 2-OA-OVA and α-GalCer exposure, i.e. before the onset of disease, significantly reduces the portal inflammatory response, production of Th1 cytokines and appearance of liver fibrosis. It also reduced the liver lymphotropic chemokines CCL5, CCL19, CXCL9, and CXCL10. Secondly, and more importantly, therapeutic use of AAV-IL-22, administered after the onset of disease, achieved a greater hurdle and significantly improved portal pathology. Further the improvements in inflammation were negatively correlated with levels of CCL5 and CXCL10 and positively correlated with levels of IL-22. In conclusion, we submit that the clinical use of IL-22 has a potential role in modulating the inflammatory portal process in patients with PBC., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The germinal center (GC) is divided into a dark zone (DZ) and a light zone (LZ). GC B cells must cycle between these zones to achieve efficient Ab affinity maturation. Follicular dendritic cells (FDCs) are well characterized for their role in supporting B cell Ag encounter in primary follicles and in the GC LZ. However, the properties of stromal cells supporting B cells in the DZ are relatively unexplored. Recent work identified a novel stromal population of Cxcl12-expressing reticular cells (CRCs) in murine GC DZs. In this article, we report that CRCs have diverse morphologies, appearing in open and closed networks, with variable distribution in lymphoid tissue GCs. CRCs are also present in splenic and peripheral lymph node primary follicles. Real-time two-photon microscopy of Peyer's patch GCs demonstrates B cells moving in close association with CRC processes. CRCs are gp38(+) with low to undetectable expression of FDC markers, but CRC-like cells in the DZ are lineage marked, along with FDCs and fibroblastic reticular cells, by CD21-Cre- and Ccl19-Cre-directed fluorescent reporters. In contrast to FDCs, CRCs do not demonstrate dependence on lymphotoxin or TNF for chemokine expression or network morphology. CRC distribution in the DZ does require CXCR4 signaling, which is necessary for GC B cells to access the DZ and likely to interact with CRC processes. Our findings establish CRCs as a major stromal cell type in the GC DZ and suggest that CRCs support critical activities of GC B cells in the DZ niche through Cxcl12 expression and direct cell-cell interactions., (Copyright © 2015 by The American Association of Immunologists, Inc.)

There is a lack of an HSV-2 vaccine, in part as the result of various factors that limit robust and long-term memory immune responses at the mucosal portals of viral entry. We previously demonstrated that chemokine CCL19 augmented mucosal and systemic immune responses to HIV-1 envelope glycoprotein. Whether such enhanced immunity can protect animals against virus infection remains to be addressed. We hypothesized that using CCL19 in a fusion form to direct an immunogen to responsive immunocytes might have an advantage over CCL19 being used in combination with an immunogen. We designed two fusion constructs, plasmid (p)gBIZCCL19 and pCCL19IZgB, by fusing CCL19 to the C- or N-terminal end of the extracellular HSV-2 glycoprotein B (gB) with a linker containing two (Gly4Ser)2 repeats and a GCN4-based isoleucine zipper motif for self-oligomerization. Following immunization in mice, pgBIZCCL19 and pCCL19IZgB induced strong gB-specific IgG and IgA in sera and vaginal fluids. The enhanced systemic and mucosal Abs showed increased neutralizing activity against HSV-2 in vitro. Measurement of gB-specific cytokines demonstrated that gB-CCL19 fusion constructs induced balanced Th1 and Th2 cellular immune responses. Moreover, mice vaccinated with fusion constructs were well protected from intravaginal lethal challenge with HSV-2. Compared with pgB and pCCL19 coimmunization, fusion constructs increased mucosal surface IgA(+) cells, as well as CCL19-responsive immunocytes in spleen and mesenteric lymph nodes. Our findings indicate that enhanced humoral and cellular immune responses can be achieved by immunization with an immunogen fused to a chemokine, providing information for the design of vaccines against mucosal infection by HSV-2 and other sexually transmitted viruses., (Copyright © 2015 by The American Association of Immunologists, Inc.)

In addition to the secretion of Ag-specific Abs, B cells may play an important role in the generation of immune responses by efficiently presenting Ag to T cells. We and other investigators recently described a subpopulation of CD11c(+) B cells (Age/autoimmune-associated B cells [ABCs]) that appear with age, during virus infections, and at the onset of some autoimmune diseases and participate in autoimmune responses by secreting autoantibodies. In this study, we assessed the ability of these cells to present Ag and activate Ag-specific T cells. We demonstrated that ABCs present Ag to T cells, in vitro and in vivo, better than do follicular B cells (FO cells). Our data indicate that ABCs express higher levels of the chemokine receptor CCR7, have higher responsiveness to CCL21 and CCL19 than do FO cells, and are localized at the T/B cell border in spleen. Using multiphoton microscopy, we show that, in vivo, CD11c(+) B cells form significantly more stable interactions with T cells than do FO cells. Together, these data identify a previously undescribed role for ABCs as potent APCs and suggest another potential mechanism by which these cells can influence immune responses and/or the development of autoimmunity., (Copyright © 2015 by The American Association of Immunologists, Inc.)

T lymphocyte migration is crucial for adaptive immunity. Manipulation of signaling molecules controlling cell migration combined with in-vitro cell migration analysis provides a powerful research approach. Microfluidic devices, which can precisely configure chemoattractant gradients and allow quantitative single cell analysis, have been increasingly applied to cell migration and chemotaxis studies. However, there are a very limited number of published studies involving microfluidic migration analysis of genetically manipulated immune cells. In this study, we describe a simple microfluidic method for quantitative analysis of T cells expressing transfected chemokine receptors and other cell migration signaling probes. Using this method, we demonstrated chemotaxis of Jurkat transfectants expressing wild-type or C-terminus mutated CCR7 within a gradient of chemokine CCL19, and characterized the difference in transfectant migration mediated by wild-type and mutant CCR7. The EGFP-tagged CCR7 allows identification of CCR7-expressing transfectants in cell migration analysis and microscopy assessment of CCR7 dynamics. Collectively, our study demonstrated the effective use of the microfluidic method for studying CCR7 mediated T cell transfectant migration. We envision this developed method will provide a useful platform to functionally test various signaling mechanisms at the cell migration level., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Antibodies are critical for defense against a variety of microbes, but they may also be pathogenic in some autoimmune diseases. Many effector functions of antibodies are mediated by Fcγ receptors (FcγRs), which are found on most immune cells, including dendritic cells (DCs)-important antigen-presenting cells that play a central role in inducing antigen-specific tolerance or immunity. Following antigen acquisition in peripheral tissues, DCs migrate to draining lymph nodes via the lymphatics to present antigen to T cells. Here we demonstrate that FcγR engagement by IgG immune complexes (ICs) stimulates DC migration from peripheral tissues to the paracortex of draining lymph nodes. In vitro, IC-stimulated mouse and human DCs showed greater directional migration in a chemokine (C-C) ligand 19 (CCL19) gradient and increased chemokine (C-C) receptor 7 (CCR7) expression. Using intravital two-photon microscopy, we observed that local administration of IC resulted in dermal DC mobilization. We confirmed that dermal DC migration to lymph nodes depended on CCR7 and increased in the absence of the inhibitory receptor FcγRIIB. These observations have relevance to autoimmunity because autoantibody-containing serum from humans with systemic lupus erythematosus (SLE) and from a mouse model of SLE also increased dermal DC migration in vivo, suggesting that this process may occur in lupus, potentially driving the inappropriate localization of autoantigen-bearing DCs.

The Wiskott-Aldrich syndrome protein (WASp) is important for actin polymerization in T cells and for their migration. WASp-interacting protein (WIP) binds to and stabilizes WASp and also interacts with actin. Cytoskeletal and functional defects are more severe in WIP(-/-) T cells, which lack WASp, than in WASp(-/-) T cells, suggesting that WIP interaction with actin may be important for T cell cytoskeletal integrity and function. We constructed mice that lack the actin-binding domain of WIP (WIPΔABD mice). WIPΔABD associated normally with WASp but not F-actin. T cells from WIPΔABD mice had normal WASp levels but decreased cellular F-actin content, a disorganized actin cytoskeleton, impaired chemotaxis, and defective homing to lymph nodes. WIPΔABD mice exhibited a T cell intrinsic defect in contact hypersensitivity and impaired responses to cutaneous challenge with protein antigen. Adoptively transferred antigen-specific CD4(+) T cells from WIPΔABD mice had decreased homing to antigen-challenged skin of wild-type recipients. These findings show that WIP binding to actin, independently of its binding to WASp, is critical for the integrity of the actin cytoskeleton in T cells and for their migration into tissues. Disruption of WIP binding to actin could be of therapeutic value in T cell-driven inflammatory diseases., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

The development of dendritic cell based vaccines is a promising approach in cancer immunotherapy. For their successful use in the clinics, the propagation and functionality of DCs is crucial. We earlier established a two-step method for the large scale generation of DCs from umbilical cord blood derived MNCs/CD34(+) cells. This work aims at improving their functionality based on the following observations: in vitro generated DCs can be less efficient in migration and other functional activities due to lower eicosanoid levels. The production of eicosanoids from Arachidonic Acid (AA) can be hampered due to suppression of the enzyme phospholipase A2 by IL-4, an essential cytokine required for the differentiation of DCs. We hypothesized that exogenous addition of AA to the culture media during DC generation may result in DCs with improved functionality. DCs were generated with and without AA. The two DC sets were compared by phenotypic analysis, morphology and functional assays like antigen uptake, MLR, CTL assay and in vitro and in vivo migration. Though there were no differences between the two types of DCs in terms of morphology, phenotype and antigen uptake, AA(+) DCs exhibited an enhanced in vitro and in vivo migration, T cell stimulatory capacity, CTL activity and significantly higher transcript levels of COX-2. AA(+) DCs also show a favorable Th1 cytokine profile than AA- DCs. Thus addition of AA to the culture media is skewing the DCs towards the secretion of more IL-12 and less of IL-10 along with the restoration of eicosanoids levels in a COX-2 mediated pathway thereby enhancing the functionality of these cells to be used as a potent cellular vaccine. Taken together, these findings will be helpful in the better contriving of DC based vaccines for cancer immunotherapy.

Since recent data suggest that nanoparticles and modified vaccinia ankara (MVA) vectors could play a pivotal role in HIV-1 therapeutics and vaccine design, in an ex vivo model of human monocyte-derived dendritic cells (MDDCs), we compared two different loading strategies with HIV-1 vaccine vehicles, either viral or synthetic derived. We used polylactic acid (PLA) colloidal biodegradable particles, coated with HIV Gag antigens (p24), and MVA expressing Gag (rMVA-gag and rMVA-gag/trans membrane) or Tat, Nef and Rev genes (rMVA tat+rev and rMVA nef). PLA-p24 captured by MDDCs from HIV-1 individuals induced a slight degree of MDDC maturation, cytokine and chemokine secretion and migration towards a gradient of CCL19 chemokine and highly increased HIV-specific CD8(+) T-cell proliferation compared with p24 alone. After complete maturation induction of PLA-p24-pulsed MDDCs, maximal migration towards a gradient of CCL19 chemokine and induction of HIV-specific T-cell proliferation (two-fold higher for CD4(+) than CD8(+)) and cytokine secretion (IFN-γ and IL-2) in the co-culture were observed. Upon exposure to MVA-gag, MDDCs produced cytokines and chemokines and maintained their capacity to migrate to a gradient of CCL19. MDDCs infected with MVA-gag and MVA-gag trans-membrane were able to induce HIV-specific CD8(+) proliferation and secretion of IFN-γ, IL-2, IL-6 and TNF-α. We conclude that both HIV antigens loading strategies (PLA-p24 nanoparticles or MVA expressing HIV genes) induce HIV-1-specific T-cell responses, which are able to kill autologous gag-expressing cells. Thus, they are plausible candidates for the development of anti-HIV vaccines., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Background: The purpose of this study was to evaluate the relationship between chemokines and dendritic cells (DCs) in human chronic periodontitis (CP)., Methods: Gingival samples were obtained from 23 individuals with CP, and six samples of normal mucosa (NM) overlapping the third molar were used to control for the chemokine levels. Periodontal examination was conducted. Immunohistochemistry was performed for Factor XIIIa(+) and cluster of differentiation (CD)1a(+) immature DCs and CD83(+) mature DCs. Levels of the CC chemokine ligand (CCL)2, CCL3, CCL5, CCL19, CCL20, and CXC chemokine ligand (CXCL)8 were measured in gingival tissues using enzyme-linked immunosorbent assay. Inflammatory infiltrate, DCs, chemokines, classification of human CP, and clinical parameters were correlated and compared., Results: The expression of CCL2 and CCL20 was positively correlated with increased densities of CD1a(+) DCs. CCL3 and CXCL8 were positively related to the clinical attachment level. CCL3, CCL5, CCL19, and CXCL8 levels increased in the gingival samples of patients with CP compared with NM, whereas CCL20 levels increased in advanced CP compared with mild-moderate CP., Conclusions: More CD1a(+) immature DCs are related to CCL2 and CCL20. CCL3 and CXCL8 chemokines are related to a greater severity of human CP.

The SRC family of kinases (SFKs) is crucial to malignant growth, but also important for signaling in immune cells such as dendritic cells (DCs). These specialized antigen-presenting cells are essential for inducing and boosting specific T-cell responses against pathogens and malignancies. Targeted therapy with SFK inhibitors holds great promise as a direct anti-cancer treatment, but potentially also as an indirect treatment via immunomodulation. Here, we investigated whether the BCR-ABL/SRC inhibitor dasatinib would modulate the major effector functions of DCs, especially their migration, a prerequisite to interaction with lymphocytes in secondary lymphoid organs. We report for the first time that dasatinib more than doubled the number of mature human monocyte-derived DCs (moDCs) migrating toward a CCL19 gradient despite unchanged CCR7 expression when used for pretreatment. These effects were caused by dephosphorylation of SFKs, as confirmed by the specific SFK inhibitor SRC inhibitor 1, leading to dephosphorylation of the inhibitory immunoreceptors Siglec-9 and Siglec-3. The specific blocking of the latter also enhanced migration and underlined the importance of these SFK-dependent receptor systems for migration of moDCs. Dasatinib hampered the secretion of interleukin-12 by moDCs at clinically relevant concentrations. In contrast, endocytosis or boosting of cytomegalovirus-specific CD8(+) T-cell responses remained unaltered when applying dasatinib-pretreated moDCs, in line with minor effects on the expression of co-stimulatory molecules essential for DC-T cell interaction. The induction of enhanced migration of moDCs may potentially be useful in chemo-immunotherapeutic applications. Thus, the use of dasatinib or blocking Siglec antibodies as adjuvants in this setting to induce stronger immune responses is worthy of further study., (Copyright © 2014 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.)

Chemokines promote T cell migration by transmitting signals that induce T cell polarization and integrin activation and adhesion. Mst1 kinase is a key signal mediator required for both of these processes; however, its molecular mechanism remains unclear. Here, we present a mouse model in which Mst1 function is disrupted by a hypomorphic mutation. Microscopic analysis of Mst1-deficient CD4 T cells revealed a necessary role for Mst1 in controlling the localization and activity of Myosin IIa, a molecular motor that moves along actin filaments. Using affinity specific LFA-1 antibodies, we identified a requirement for Myosin IIa-dependent contraction in the precise spatial distribution of low and higher affinity LFA-1 on the membrane of migrating T cells. Mst1 deficiency or Myosin inhibition resulted in multipolar cells, difficulties in uropod detachment and mis-localization of low affinity LFA-1. Thus, Mst1 regulates Myosin IIa dynamics to organize high and low affinity LFA-1 to the anterior and posterior membrane during T cell migration.

The chemokine receptor CCR7 plays a crucial role in the homing of central memory and naive T cells to peripheral lymphoid organs. Here, we show that the HIV-1 accessory protein Vpu downregulates CCR7 on the surface of CD4(+) T cells. Vpu and CCR7 were found to specifically interact and colocalize within the trans-Golgi network, where CCR7 is retained. Downmodulation of CCR7 did not involve degradation or endocytosis and was strictly dependent on Vpu expression. Stimulation of HIV-1-infected primary CD4(+) T cells with the CCR7 ligand CCL19 resulted in reduced mobilization of Ca(2+), reduced phosphorylation of Erk1/2, and impaired migration toward CCL19. Specific amino acid residues within the transmembrane domain of Vpu that were previously shown to be critical for BST-2 downmodulation (A14, A18, and W22) were also necessary for CCR7 downregulation. These results suggest that BST-2 and CCR7 may be downregulated via similar mechanisms., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Cell therapy regimens are frequently compromised by low-efficiency cell homing to therapeutic niches. Improvements in this regard would enhance effectiveness of clinically applicable cell therapy. The major regulators of tissue-specific cellular migration are chemokines, and therefore selection of therapeutic cellular populations for appropriate chemokine receptor expression would enhance tissue-homing competence. A number of practical considerations preclude the use of Abs in this context, and alternative approaches are required. In this study, we demonstrate that appropriately labeled chemokines are at least as effective in detecting their cognate receptors as commercially available Abs. We also demonstrate the utility of biotinylated chemokines as cell-sorting reagents. Specifically, we demonstrate, in the context of CCR7 (essential for lymph node homing of leukocytes), the ability of biotinylated CCL19 with magnetic bead sorting to enrich for CCR7-expressing cells. The sorted cells demonstrate improved CCR7 responsiveness and lymph node-homing capability, and the sorting is effective for both T cells and dendritic cells. Importantly, the ability of chemokines to detect CCR7, and sort for CCR7 positivity, crosses species being effective on murine and human cells. This novel approach to cell sorting is therefore inexpensive, versatile, and applicable to numerous cell therapy contexts. We propose that this represents a significant technological advance with important therapeutic implications., (Copyright © 2014 by The American Association of Immunologists, Inc.)

Polyphenols have been shown to exhibit anti-inflammatory, anti-oxidant and immunomodulatory activities. However, the effects of anthocyanins, flavonoids of great nutritional interest, in particular of their metabolite protocatechuic acid (PCA) on the phenotypic and functional maturation of human dendritic cells (DCs) are still largely unknown. In this study, we report that PCA is efficiently taken up and accumulated in human monocyte-derived DCs (MD-DCs). PCA exposure of MD-DCs markedly impaired the production of proinflammatory cytokines and chemokines (i.e. IL-6, IL-8 and CCL2) in response to bacterial endotoxin and leptin, and down-regulated the lipopolysaccharide (LPS)-induced migratory response of MD-DCs to CCL19. Conversely, the phenotypic profile induced by LPS-mediated activation as well as IL-12 production was not affected. Interestingly, we found that PPARγ is a main factor in the PCA-induced effects as blocking its activity abolish PCA capacity to down-regulate IL-6 and IL-8, but not CCL2, secretion and to inhibit MD-DC migration. In keeping with this observation, cytosol to nucleus translocation and PPARγ activity were found to be directly stimulated by PCA exposure of MD-DCs. These novel findings provide new insight into the immunoregulatory effects of polyphenol metabolites in DCs opening new perspectives on their potential application in the prevention of acute and chronic inflammatory diseases., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

Microbial pathogens are able to modulate host cells and evade the immune system by multiple mechanisms. For example, Salmonella injects effector proteins into host cells and evades the host immune system in part by inhibiting dendritic cell (DC) migration. The identification of microbial factors that modulate normal host functions should lead to the development of new classes of therapeutics that target these pathways. Current screening methods to identify either host or pathogen genes involved in modulating migration towards a chemical signal are limited because they do not employ stable, precisely controlled chemical gradients. Here, we develop a positive selection microfluidic-based genetic screen that allows us to identify Salmonella virulence factors that manipulate DC migration within stable, linear chemokine gradients. Our screen identified 7 Salmonella effectors (SseF, SifA, SspH2, SlrP, PipB2, SpiC and SseI) that inhibit DC chemotaxis toward CCL19. This method is widely applicable for identifying novel microbial factors that influence normal host cell chemotaxis as well as revealing new mammalian genes involved in directed cell migration.

Aims: The homeostatic chemokines, CCL19 and CCL21 and their receptor CCR7, have recently been linked to atherogenesis. We investigated the expression of CCL19/CCL21/CCR7 in carotid atherosclerosis as well as the ability of these chemokines to modulate lipid accumulation in macrophages and vascular smooth muscle cell (SMC) phenotype., Methods and Results: Our major findings were: (i) patients with carotid atherosclerosis (n = 158) had increased plasma levels of CCL21, but not of CCL19, compared with controls (n = 20), with particularly high levels in symptomatic (n = 99) when compared with asymptomatic (n = 59) disease. (ii) Carotid plaques showed markedly increased mRNA levels of CCL21 and CCL19 in symptomatic (n = 14) when compared with asymptomatic (n = 7) patients, with CCR7 localized to macrophages and vascular SMC (immunohistochemistry). (iii) In vitro, CCL21, but not CCL19, increased the binding of modified LDL and promoted lipid accumulation in THP-1 macrophages. (iv) CCL19, but not CCL21, increased proliferation and release and activity of matrix metalloproteinase (MMP) 1 in vascular SMC. (v) The differential effects of CCL19 and CCL21 in macrophages and SMC seem to be attributable to divergent signalling pathways, with CCL19-mediated activation of AKT in SMC- and CCL21-mediated activation of extracellular signal-regulated kinase 1/2 in macrophages., Conclusion: CCL19 and CCL21 are up-regulated in carotid atherosclerosis. The ability of CCL21 to promote lipid accumulation in macrophages and of CCL19 to induce proliferation and MMP-1 expression in vascular SMC could contribute to their pro-atherogenic potential.

Human endogenous retroviruses (HERV) are associated with many diseases such as autoimmune diseases and cancer. Although the frequent expression of a variety of HERVs in tumor cells has been demonstrated, their functional contributions in cancer are as yet unclear. Intriguingly, HERVs and other retroviruses include an immunosuppressive domain in their transmembrane envelope proteins, but its mechanism of action and cancer relevance are obscure. In this study, we demonstrate that the human endogenous retrovirus HERV-H has a critical role in tumor metastasis and immune escape. We found that expression of herv-h mRNA was elevated in metastatic tumor cells undergoing epithelial-to-mesenchymal transition (EMT) and in primary tumor tissues from advanced colon cancer. The immunosuppressive peptide H17 derived from HERV-H was sufficient to induce EMT in tumor cells that expressed low levels of HERV-H, and it amplified this event within the tumor microenvironment. H17 also stimulated CCL19 expression in tumor cells, which in turn recruited and expanded a population of pluripotent immunoregulatory CD271(+) cells, which included mesenchymal stem cells and myeloid-derived suppressor cells. In tumor tissues from patients with advanced colon cancer, we confirmed that CD271(+) cells were increased in HERV-H(+)CCL19(+) tumor tissues. Notably, RNAi-mediated change of HERV-H or CCL19, or depletion of CD271(+) cells, improved immune responses in vitro and in vivo accompanied by tumor regression. Together, our results argued that HERV-H is a critical determinant of immune escape in cancer, suggesting its candidacy as a promising therapeutic target to treat patients with advanced cancer., (©2014 AACR)

Chemokines are classified into several different subfamilies, of which CC chemokines constitute the largest subfamily in teleost. The prominent structural characteristic of CC chemokines is the presence of an Asp-Cys-Cys-Leu (DCCL) motif. To date, cDNA sequences of several CC chemokines have been identified in turbot (Scophthalmus maximus), however, the activity and function of these putative chemokines remain unknown. In this study, we examined the biological effect of the turbot CC chemokine SmCCL19, which has been previously reported as KC70 and shown to be regulated in expression by bacterial infection. To facilitate functional analysis, recombinant SmCCL19 (rSmCCL19) and a mutant form of SmCCL19, SmCCL19M, that bears serine substitutions at the two cysteine residues of the DCCL motif were purified from Escherichia coli. Chemotactic analysis showed that rSmCCL19 induced migration of head kidney leukocytes in a dose-dependent manner, whereas rSmCCL19M caused no apparent cellular migration. To examine the in vivo effect of rSmCCL19, turbot were administered with rSmCCL19 or rSmCCL19M before being inoculated with viral and bacterial pathogens. Subsequent tissue infection analysis showed that the viral and bacterial loads in rSmCCL19-adminsitered fish were significantly reduced, whereas the pathogen loads in rSmCCL19M-adminsitered fish were largely comparable to those in the control fish. Consistent with these observations, significant inductions of immune relevant genes were observed in rSmCCL19-adminsitered fish but not in rSmCCL19M-adminsitered fish. Taken together, these results indicate that SmCCL19 recruits leukocytes and augments host immune defense in a manner that depends on the conserved DCCL motif., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Human eosinophils display directed chemotactic activity toward an array of soluble chemokines. Eosinophils have been observed to migrate to draining lymph nodes in experimental models of allergic inflammation, yet it is unknown whether eosinophils express CCR7, a key chemokine receptor in coordinating leukocyte trafficking to lymph nodes. The purpose of this study is to demonstrate expression of CCR7 by human eosinophils and functional responses to CCL19 and CCL21, the known ligands of CCR7. Human eosinophils were purified by negative selection from healthy donors. CCR7 expression of freshly purified, unstimulated eosinophils and of IL-5-primed eosinophils was determined by flow cytometry and Western blot. Chemotaxis to CCL19 and CCL21 was measured in transwell assays. Shape changes to CCL19 and CCL21 were analyzed by flow cytometry and microscopy. Calcium fluxes of fluo-4 AM-loaded eosinophils were recorded by flow cytometry after chemokine stimulation. ERK phosphorylation of CCL19- and CCL21-stimulated eosinophils was measured by Western blot and Luminex assay. Human eosinophils expressed CCR7 as demonstrated by flow cytometry and Western blots. Eosinophils exhibited detectable cell surface expression of CCR7. IL-5-primed eosinophils exhibited chemotaxis toward CCL19 and CCL21 in a dose-dependent fashion. Upon stimulation with CCL19 or CCL21, IL-5-primed eosinophils demonstrated dose-dependent shape changes with polarization of F-actin and exhibited calcium influxes. Finally, primed eosinophils stimulated with CCL19 or CCL21 exhibited increased phosphorylation of ERK in response to both CCR7 ligands. We demonstrate that human eosinophils express CCR7 and have multipotent responses to the known ligands of CCR7.

Here, we report on the successful programming of dendritic cells (DCs) using selectively applied mixtures of chemokines as a novel protocol for engineering vaccine efficiency. Antigen internalization by DCs is a pivotal step in antigen uptake/presentation for bridging innate and adaptive immunity and in exogenous gene delivery used in vaccine strategies. Contrary to most approaches to improve vaccine efficiency, active enhancement of antigen internalization by DCs as a vaccine strategy has been less studied because DCs naturally down-regulate antigen internalization upon maturation. Whereas chemokines are mainly known as signal proteins that induce leucocyte chemotaxis, very little research has been carried out to identify any additional effects of chemokines on DCs following maturation. Here, immature DCs are pre-treated with select chemokines before intentional maturation using lipopolysaccharide (LPS). When pre-treated with a mixture of CCL3 and CCL19 in a 7 : 3 ratio, then matured with LPS, chemokine pre-treated DCs exhibited 36% higher antigen uptake capacity than immature DCs and 27% higher antigen-processing capacity than immature DCs treated only with LPS. Further, CCL3 : CCL19 (7 : 3) pre-treatment of DCs modulated MHC molecule expression and secretion of various cytokines of DCs. Collectively, DC programming was feasible using a specific chemokine combination and these results provide a novel strategy for enhancing DC-based vaccine efficiency. In Part II, we report on the phenotype changes and antigen presentation capacity of chemokine pre-treated murine bone marrow-derived DCs examined in long-term co-culture with antigen-specific CD4(+) T cells., (© 2012 Blackwell Publishing Ltd.)

In a companion article to this study,(1) the successful programming of a JAWSII dendritic cell (DC) line's antigen uptake and processing was demonstrated based on pre-treatment of DCs with a specific 'cocktail' of select chemokines. Chemokine pre-treatment modulated cytokine production before and after DC maturation [by lipopolysaccharide (LPS)]. After DC maturation, it induced an antigen uptake and processing capacity at levels 36% and 82% higher than in immature DCs, respectively. Such programming proffers a potential new approach to enhance vaccine efficiency. Unfortunately, simply enhancing antigen uptake does not guarantee the desired activation and proliferation of lymphocytes, e.g. CD4(+) T cells. In this study, phenotype changes and antigen presentation capacity of chemokine pre-treated murine bone marrow-derived DCs were examined in long-term co-culture with antigen-specific CD4(+) T cells to quantify how chemokine pre-treatment may impact the adaptive immune response. When a model antigen, ovalbumin (OVA), was added after intentional LPS maturation of chemokine-treated DCs, OVA-biased CD4(+) T-cell proliferation was initiated from ~ 100% more undivided naive T cells as compared to DCs treated only with LPS. Secretion of the cytokines interferon-γ, interleukin-1β, interleukin-2 and interleukin-10 in the CD4(+) T cell : DC co-culture (with or without chemokine pre-treatment) were essentially the same. Chemokine programming of DCs with a 7 : 3 ratio of CCL3 : CCL19 followed by LPS treatment maintained partial immature phenotypes of DCs, as indicated by surface marker (CD80 and CD86) expression over time. Results here and in our companion paper suggest that chemokine programming of DCs may provide a novel immunotherapy strategy to obviate the natural endocytosis limit of DC antigen uptake, thus potentially increasing DC-based vaccine efficiency., (© 2012 Blackwell Publishing Ltd.)