Purpose: T cell recruitment, survival and proliferation are the important limitations to chimeric antigen receptor (CAR) T cells therapy in the treatment of solid tumors. In this study we engineered CAR-T cells to co-express cytokines IL-7 and CCL21 (7×21 CAR-T), a cytokine combination in order to improve proliferation and chemotaxis of CAR-T cells.

Experimental design: CLDN18.2 specific second-generation CAR-T cells coexpressing cytokines were prepared using retroviral vector transduction. The proliferation and migration of genetically engineered CAR-T cells were evaluated in vitro. The antitumor activities of genetically engineered CAR-T cells were evaluated against multiple solid tumors in C57BL/6 mice in vivo.

Results: In vitro, the proliferation and chemotaxis of 7×21 CAR-T cells are significantly improved when compared to those of the conventional CAR-T cells. In vivo, 7×21 CAR-T cells revealed superior therapeutic effects to either conventional CAR-T cells or 7×19 CAR-T cells which co-expressing IL-7 and CCL19 as previous reported in three different solid tumors without cyclophosphamide (CPA) precondition. Interestingly, 7×21 CAR-T cells could also suppress the tumor growth with heterogenous antigen expression and even induce tumor complete remission. Mechanistically, IL-7 and CCL21 significantly improved survival and infiltration of CAR-T cells and DCs in tumor. Additionally, CCL21 also inhibited the tumor angiogenesis as proved by immunohistochemistry.

Conclusion: Coexpression of IL-7 and CCL21 could boost CAR-T cells antitumor activity and 7×21 CAR-T cells may be served as a promising therapy strategy for solid tumors.

Knowledge of the molecules used by the maternal reproductive tract to regulate development of the preimplantation embryo is largely incomplete. The goal of the present experiment was to identify candidates for this function. The approach was to assess expression patterns in the endometrium and oviduct of 93 genes encoding for hormones, growth factors, chemokines, cytokines, and WNT-related molecules. Results show that all of the genes were expressed in the reproductive tract. Expression in oviduct was affected by day of the estrous cycle for 21 genes with 11 genes having highest expression at estrus (CCL21, CTGF, CXCL10, CXCL16, DKK3, FGF10, IL18, IL33, IL34, PGF, and SFRP2), 1 gene at d 3 (WNT4), 8 at d 5 (BMP7, HGF, IL6, SFRP1, TGFB1, WIF1, WNT2, and WNT5A), and 1 at d 7 (IK). For endometrium, expression of 34 genes was affected by day of the estrous cycle with 11 having highest expression at d 0 (BMP7, CCL14, CCL21, CCL26, CTGF, CXCL12, IGF2, IL16, IL33, SFRP2, and WIF1), 2 at d 3 (HDGF, IL15), 14 at d 5 (CSF2, CX3CL1, CXCL3, FGF1, FGF2, GRO1, HGF, IGF1, IL1B, IL8, SFRP1, SFRP4, WNT5A, and WNT16), and 7 at d 7 (CXCL16, FGF13, HDGFRP2, TDGF1, VEGFB, WNT7A, and WNT11). Results are consistent with a set of genes regulated by estradiol early in the estrous cycle and another set regulated by progesterone later in the cycle. The cell-signaling genes identified here as being expressed in the oviduct and endometrium could serve to regulate early embryonic development in a stage-of-pregnancy-specific manner.

BACKGROUND

Adherence of human mesangial cells to the surrounding matrix contributes to glomerular homeostasis and is important for the maintenance of glomerular architecture and function in normal adult human kidney. The expression of chemokines and corresponding chemokine receptors on adjacent intrinsic renal cells indicates a novel chemokine/chemokine receptor function on nonimmune cells important for glomerular homeostasis. A constitutive expression of the chemokine SLC/CCL21 on human podocytes and of its corresponding receptor CCR7 on mesangial cells was shown before. SLC/CCL21 has a positive effect on proliferation and migration of mesangial cells and leads to increased cell survival in Fas-induced apoptosis. In leukocytes chemokines mediate integrin-dependent firm adhesion. Therefore, we examined the influence of chemokine receptor CCR7 activation by SLC/CCL21 on adhesive properties of human mesangial cells to matrix molecules.

METHODS

Adhesion assays, mechanical detachment assays, and evaluation of integrin activation by integrin-linked kinase activity were performed. Changes in the cytoskeletal F-actin were illustrated by phalloidin immunofluorescence staining.

RESULTS

SLC/CCL21 stimulation enhanced adhesiveness to fibronectin in a time- and concentration-dependent manner. SLC/CCL21 also increased the firmness of mesangial cells adhesion as judged by detachment assays. Furthermore activation of integrin-linked kinase occurred with SLC/CCL21 addition to mesangial cells, resulting in increased phosphorylation of glycogen synthase kinase-3 (GSK-3) and protein kinase B (PKB/Akt). Exposure of mesangial cells to SLC/CCL21 also resulted in F-actin rearrangements with membrane ruffling and extensions leading to bridging between mesangial cells.

CONCLUSIONS

Activation of CCR7 on mesangial cells by SLC/CCL21 enhances the degree and firmness of cell adhesion and increases cell spreading and the formation of cell-cell contacts. This includes integrin-linked kinase activation and F-actin rearrangements. Thus, local chemokine generation and chemokine receptor expression on mesangial cells may play an important role in the maintenance of glomerular homeostasis and in local remodeling processes.

Type 1 diabetes (T1D) is a T cell-mediated autoimmune disease characterized by the destruction of insulin-producing β cells in the pancreatic islets. The migration of T cells from blood vessels into pancreas is critical for the development of islet inflammation and β cell destruction in T1D. To define the roles of C-C chemokine receptor type 7 (CCR7) in recruitment of T cells into islets, we used laser capture microdissection to isolate tissue from inflamed islets of nonobese diabetic (NOD) mice and uninflamed islets of BALB/c and young NOD mice. RT-PCR analyses detected mRNAs for CCR7 and its chemokine ligands CCL19 (ELC; MIP-3β) and CCL21 (SLC) in captures from inflamed, but not from uninflamed, islets. Immunohistology studies revealed that high endothelial venules in inflamed islets co-express CCL21 protein and MAdCAM-1 (an adhesion molecule that recruits lymphocytes into islets). Desensitization of lymphocyte CCR7 blocked about 75 % of T cell migration from the bloodstream into inflamed islets, but had no effect on B cell migration into islets. These results indicate that CCR7 and its ligands are important in the recruitment of T cells into inflamed islets and thus in the pathogenesis of T1D.

The time course and cellular basis for inflammation-induced hypertrophy of adipose tissue were investigated over 20 weeks in mature male rats. Mild inflammation was induced by subcutaneous injection of 20 microg lipopolysaccharide into one hind-leg three times/week for 4 or 8 weeks, followed by up to 12 weeks 'rest' without intervention. Mean volume and frequency of apoptosis (TUNEL assay) were measured in adipocytes isolated from sites defined by their anatomical relations to lymph nodes, plus numbers of CCL21-stimulated lymph node-derived and adipose tissue-derived dendritic cells. Experimental inflammation increased dendritic cells and adipocyte apoptosis in the locally stimulated popliteal depot and the lymphoid tissue-associated regions of the contralateral popliteal and mesentery and omentum. Responses declined slowly after inflammation ended, but all measurements from the locally stimulated popliteal depot, and the omentum, were still significantly different from controls after 12 weeks rest. The locally stimulated popliteal adipose tissue enlarged by 5% within 4 weeks and remained larger than the control. We conclude that prolonged inflammation induces permanent enlargement, greater adipocyte turnover and increased dendritic cell surveillance in the adjacent adipose tissue and the omentum. The experiment suggests a mechanism for selective hypertrophy of lymphoid tissue-associated adipose tissue in chronic stress and inflammatory disorders, including impaired lymph drainage, Crohn's disease and HIV-associated lipodystrophy, and a link between evolutionary fitness, sexual selection and aesthetically pleasing body symmetry. It would be useful for further study of molecular mechanisms in inflammation-induced local hypertrophy of adipose tissue and development of specific therapies that avoid interference with whole-body lipid metabolism.

In mammals, CCR7 is the chemokine receptor for the CCL19 and CCL21 chemokines, molecules with a major role in the recruitment of lymphocytes to lymph nodes and Peyer's patches in the intestinal mucosa, especially naïve T lymphocytes. In the current work, we have identified a CCR7 orthologue in rainbow trout (Oncorhynchus mykiss) that shares many of the conserved features of mammalian CCR7. The receptor is constitutively transcribed in the gills, hindgut, spleen, thymus and gonad. When leukocyte populations were isolated, IgM(+) cells, T cells and myeloid cells from head kidney transcribed the CCR7 gene. In blood, both IgM(+) and IgT(+) B cells and myeloid cells but not T lymphocytes were transcribing CCR7, whereas in the spleen, CCR7 mRNA expression was strongly detected in T lymphocytes. In response to infection with viral hemorrhagic septicemia virus (VHSV), CCR7 transcription was down-regulated in spleen and head kidney upon intraperitoneal infection, whereas upon bath infection, CCR7 was up-regulated in gills but remained undetected in the fin bases, the main site of virus entry. Concerning its regulation in the intestinal mucosa, the ex vivo stimulation of hindgut segments with Poly I:C or inactivated bacteria significantly increased CCR7 transcription, while in the context of an infection with Ceratomyxa shasta, the levels of transcription of CCR7 in both IgM(+) and IgT(+) cells from the gut were dramatically increased. All these data suggest that CCR7 plays an important role in lymphocyte trafficking during rainbow trout infections, in which CCR7 appears to be implicated in the recruitment of B lymphocytes into the gut.

Subsets of gammadelta T cells localize to distinct tissue sites in the absence of exogenous Ag stimulation or development of effector/memory cells. Selective lymphocyte homing from the blood into tissues is controlled by a multistep process involving vascular and lymphocyte adhesion molecules, and G protein-linked chemokine receptors. The role of these mechanisms in the tissue tropism of gammadelta T cells is still poorly understood. In this study, we demonstrate that a subset of gammadelta T cells, most of which express an antigenically distinct TCR and are characterized by coexpression of CD8, selectively accumulated in tissues that expressed high levels of the mucosal vascular addressin, mucosal addressin cell adhesion molecule 1. These cells expressed higher levels of alpha(4)beta(7) integrins than other gammadelta T cell subsets and selectively migrated to the CCR7 ligand secondary lymphoid-tissue chemokine (CCL21). Integrin activation by CCL21 selectively increased CD8(+)gammadelta T cell binding to recombinant mucosal addressin cell adhesion molecule 1. These results suggest that the tropism of circulating CD8(+)gammadelta T cells for mucosal tissues is due, at least in part, to selective developmental expression of adhesion molecules and chemokine receptors.

Chemokine-mediated signalling involves the activation of a Janus kinase (Jak) pathway. We have previously shown that Jak3 mediates CCR9 and CXCR4 signalling in response to CCL25 and CXCL12 in BM progenitors and thymocytes. The lack of peripheral lymph nodes and Peyer's patches observed in Jak3(-/-) mice suggested a possible role of Jak3 in CCR7-mediated homing to these organs. Here, we demonstrate phosphorylation of Jak3 in peripheral lymphocytes in response CCL19 and CCL21. In addition, Jak3(-/-) naïve T cells and pharmacologically inhibited Jak3(+/+) T lymphocytes have impaired chemotactic responses towards these ligands. Interestingly, CCR7 expression was higher in Jak3(-/-) thymocytes compared to their Jak3(+)(/-) littermates, indicating that the impaired migration must be caused by impaired CCR7-mediated signalling, in the absence of Jak3. In addition, adoptive transfer experiments showed that Jak3(+/+) mice reconstituted with Jak3(-/-) green fluorescent protein (GFP)(+) bone marrow progenitors had reduced T-lymphocyte homing to peripheral and mesenteric lymph nodes, compared to reconstitution with Jak3(+/+) GFP(+) progenitors. Furthermore, reciprocal transfer experiments indicated that Jak3(-/-) stromal cells were not responsible for the deficient T-cell homing. Finally, we performed direct competitive homing assays and demonstrated that Jak3(-/-) T lymphocytes have a clear defect in homing to peripheral and mesenteric lymph nodes, while migration to spleen was moderately impaired. Our data demonstrates that Jak3(-/-) T lymphocytes have an intrinsic defect in CCR7-mediated homing to peripheral lymphoid organs.

The identification of chemokines has profoundly changed the way we interpret the immune response, elucidating the mechanism by which inflammatory cells are recruited to the site of infection by local secretion of chemoattractants such as CXC chemokine ligand 8 (CXCL8)/interleukin-8, chemokine ligand 2 (CCL2)/monocyte chemoattractant protein 1. This novel view of the immune response has been remodeled further following observations that lymphoid tissue development derives from the coordinated secretion of homeostatic chemokines such as CCL19, CCL21, and CXCL13, which mediate recruitment and clustering of the cells involved in lymphoid organogenesis. The study of primary immunodeficiencies has demonstrated that the number of circulating leukocytes is dependent on migration amongst bone marrow, blood circulation, and inflamed tissues. Defects of leukocyte adhesion and chemotaxis as a result of mutations of beta2-integrins lead to abnormal leukocytosis and susceptibility to skin infections, as observed in leukocyte adhesion deficiency. Conversely, neutropenia in children with myelokathexis is a result of leukocyte retention in the bone marrow because of the mutations of CXC chemokine receptor 4, which affect the capacity of cells to recirculate between blood and bone marrow. Moreover, the identification of the genetic basis of primary immunodeficiencies has shown that many primary immunodeficiencies such as Wiskott-Aldrich syndrome and common variable immunodeficiencies are characterized by altered migration of leukocytes and/or disregulation of cellular response to chemokines. This paper will be focused on the interpretation of primary immunodeficiencies as defects in leukocyte circulation between blood and primary and secondary organs.

Dendritic cells (DCs) play a major role in development of cell-mediated immunotherapy due to their unique role in linking innate and adaptive immunities. In spite of improvement in this area, strategies employing ex vivo generated DCs have shown limited efficacy in clinical trials. Dendrimers have been proposed as new carriers for drug delivery in aim to ameliorate DCs antigen loading that is a pivotal point in DCs approaches. In this study, we have investigated the phenotypic and functional characteristics of human monocytes-derived dendritic cells after HIV-derived peptides uptake in vitro. We have found that iDCs and mDCs were able to capture efficiently water soluble carbosilane (CBS) dendrimer 2 G-NN16 and did not induce changes in maturation markers levels at the DCs surface. Therefore, CBS 2 G-NN16-loaded mDCs migrated as efficiently as unloaded DCs towards CCL19 or CCL21. Furthermore, DCs viability, activation of allogenic naïve CD4 + T cells by mDCs and secretion of cytokines were not significantly changed by 2 G-NN16 loading. Summing up, our data indicate that CBS 2 G-NN16 has no negative effects on the pivotal properties of DCs in vitro. It should therefore be feasible to further develop this antigen loading strategy for clinical use in immunotherapy against viral infections.

The thymus provides a microenvironment that induces the differentiation of T-progenitor cells into functional T cells and that establishes a diverse yet self-tolerant T-cell repertoire. However, the mechanisms that lead to the development of the thymus are incompletely understood. We report herein the results of screening for genes that are expressed in the third pharyngeal pouch, which contains thymic primordium. Polymerase chain reaction (PCR)-based cDNA subtraction screening for genes expressed in microdissected tissues of the third pharyngeal pouch rather than the second pharyngeal arch yielded one transcription factor, MafB, which was predominantly expressed in CD45(-)IA(-)PDGFRalpha(+) mesenchymal cells and was detectable even in the third pharyngeal pouch of FoxN1-deficient nude mice. Interestingly, the number of CD45(+) cells that initially accumulated in the embryonic thymus was significantly decreased in MafB-deficient mice. Alterations of gene expression in the embryonic thymi of MafB-deficient mice included the reduced expression of Wnt3 and BMP4 in mesenchymal cells and of CCL21 and CCL25 in epithelial cells. These results suggest that MafB expressed in third pharyngeal pouch mesenchymal cells critically regulates lymphocyte accumulation in the embryonic thymus.

OBJECTIVE

Little is known about the pathogenic mechanisms of autoimmune pancreatitis (AIP), an increasingly recognized, immune-mediated form of chronic pancreatitis. Current treatment options are limited and disease relapse is frequent. We investigated factors that contribute to the development of AIP and new therapeutic strategies.

METHODS

We used quantitative polymerase chain reaction, immunohistochemical, and enzyme-linked immunosorbent analyses to measure the expression of cytokines and chemokines in tissue and serum samples from patients with and without AIP. We created a mouse model of human AIP by overexpressing lymphotoxin (LT)α and β specifically in acinar cells (Ela1-LTab mice).

RESULTS

Messenger RNA levels of LTα and β were increased in pancreatic tissues from patients with AIP, compared with controls, and expression of chemokines (CXCL13, CCL19, CCL21, CCL1, and B-cell-activating factor) was increased in pancreatic and serum samples from patients. Up-regulation of these factors was not affected by corticosteroid treatment. Acinar-specific overexpression of LTαβ (Ela1-LTαβ) in mice led to an autoimmune disorder with various features of AIP. Chronic inflammation developed only in the pancreas but was sufficient to cause systemic autoimmunity. Acinar-specific overexpression of LTαβ did not cause autoimmunity in mice without lymphocytes (Ela1-LTab/Rag1(-/-)); moreover, lack of proinflammatory monocytes (Ela1-LTab/Ccr2(-/-)) failed to prevent AIP but prevented early pancreatic tissue damage. Administration of corticosteroids reduced pancreatitis but did not affect production of autoantibodies, such as antipancreatic secretory trypsin inhibitor in Ela1-LTab mice. In contrast, inhibition of LTβR signaling reduced chemokine expression, renal immune-complex deposition, and features of AIP in Ela1-LTab mice.

CONCLUSIONS

Overexpression of LTαβ specifically in acinar cells of mice causes features of AIP. Reagents that neutralize LTβR ligands might be used to treat patients with AIP.

Secondary lymphoid organs (SLOs) provide a niche for the initiation and regulation of T-cell responses, but the mechanisms have been poorly understood. We investigated the influence of chemokines CCL19 and CCL21 constitutively expressed in SLOs on activation-induced cell death (AICD) of CD4+ T cells. When paucity of lymph node T cells (plt) mutant mice lacking expression of CCL19/CCL21 were primed with OVA/CFA, both expansion of OVA-responding CD4+ T cells in the draining lymph nodes and an in vitro recall response were prolonged as compared with responses in wild-type (WT) mice. The apoptotic cell frequency among OVA-responding CD4+ T cells was similarly low in plt/plt and WT mice during the clonal expansion phase. However, during the clonal contraction phase, the frequency never increased in plt/plt mice, whereas in WT mice it continuously increased to a peak 18 days after immunization. The presence of CCL19/CCL21 during the in vitro stimulation of CD4+ T cells with anti-CD3 plus anti-CD28 significantly enhanced in vitro AICD induction of the restimulated T cells, partially through enhancing expression of Fas ligand. Our results suggest that CCL19/CCL21 produced by stromal cells and antigen-presenting cells regulate CD4+ T-cell immune responses in SLOs by promoting AICD.

Graves' disease (GD) is a chronic autoimmune process characterized by the production of auto-antibodies that presumably consequent to the lymphocytic infiltrates in the thyroid. Chemokine (C-C motif) ligand 21 (CCL21) is important for the circulation of CC-chemokine receptor 7 (CCR7)-expressing cells. Meanwhile, osteopontin (OPN) enhances the production of proinflammatory cytokines and chemokines through NF-κB and MAPK signaling pathways in GD. Although CCL21 has been reported to play a vital role in several autoimmune diseases, little is known about the relationship between CCL21 and GD development. This study aimed to detect the CCL21 level in GD and to examine the role of OPN in regulating CCL21 production. 40 initial GD patients, 15 euthyroid GD patients, 12 TRAb-negative GD patients, and 25 healthy control donors were recruited. CCL21 levels in plasma and culture supernatants were quantified by enzyme-linked immunosorbent assay (ELISA). CD4+ T cells were isolated from peripheral blood mononuclear cells using antibody-coated magnetic beads. Quantitative polymerase chain reaction was used to determine CCL21 expression levels in CD4+ T cells. We demonstrated for the first time that plasma CCL21 levels were overexpressed in GD patients and recovered in TRAb-negative GD patients. Moreover, CCL21 levels correlated with TRAb levels and plasma OPN concentrations. Furthermore, we demonstrated that recombinant OPN increased the expression of CCL21 in a dose- and time-dependent manner. These data indicated a clinical correlation between plasma CCL21 levels and GD. CCL21 could serve as a novel biomarker for GD as well as a potential target for TRAb-positive GD treatment.

Usual type VIN is a premalignant disorder caused by persistent HPV infection. High prevalence of VIN in immuno-suppressed women suggests that a good innate and adaptive immune response is important for defense against HPV. Here, we explored expression levels of chemokines and related these to the presence or absence of immuno-competent cells (dendritic and T-cells) in affected (HPV-positive VIN) and non-affected (HPV-negative) vulvar tissues from the same patients. Combining microarray data with quantitative real-time RT-PCR, it was observed that several important chemokines were differentially expressed between VIN and control samples (up-regulation of IL8, CXCL10, CCL20 and CCL22 and down-regulation of CXCL12, CCL21 and CCL14). Furthermore, an increased number of mature dendritic cells (CD208+) seemed to be bottled up in the dermis, and although a T-cell response (increased CD4+ and CD8+ cells) was observed in VIN, a much larger response is required to clear the infection. In summary, it seems that most mature dendritic cells do not receive the proper chemokine signal for migration and will stay in the dermis, not able to present viral antigen to naive T-cells in the lymph node. Consequently the adaptive immune response diminishes, resulting in a persistent HPV infection with increased risk for neoplasia.

High activity of dendritic cells (DCs) in inducing cytotoxic T cells (CTLs) led to their application as therapeutic cancer vaccines. The ability of DCs to produce IL-12p70 is one of the key requirements for effective CTL induction and a predictive marker of their therapeutic efficacy in vivo. We have previously reported that defined cocktails of cytokines, involving TNFα and IFNγ, induce mature type-1 polarized DCs (DC1s) which produce strongly elevated levels of IL-12 and CXCL10/IP10 upon CD40 ligation compared to "standard" PGE₂-matured DCs (sDCs; matured with IL-1β, IL-6, TNFα, and PGE₂) and show higher CTL-inducing activity. Guided by our observations that DC1s can be induced by TNFα- and IFNγ-producing CD8⁺ T cells, we have tested the feasibility of using lymphocytes to generate DC1s in a clinically-compatible process, to limit the need for clinical-grade recombinant cytokines and the associated costs. CD3/CD28 activation of bulk lymphocytes expanded them and primed them for effective production of IFNγ and TNFα following restimulation. Restimulated lymphocytes, or their culture supernatants, enhanced the maturation status of immature (i)DCs, elevating their expression of CD80, CD83 and CCR7, and the ability to produce IL-12p70 and CXCL10 upon subsequent CD40 ligation. The "lymphocyte-matured" DC1s showed elevated migration in response to the lymph-node-directing chemokine, CCL21, when compared to iDCs. When loaded with antigenic peptides, supernatant-matured DCs induced much high levels of CTLs recognizing tumor-associated antigenic epitope, than PGE₂-matured DCs from the same donors. These results demonstrate the feasibility of generation of polarized DC1s using autologous lymphocytes.

Human telomerase reverse transcriptase (TERT) has been considered a potential tumor-associated antigen for active-specific immunotherapy. However, effective specific tumor antigen-specific immunity has been difficult to induce consistently by various TERT vaccine formulations. New adjuvant strategies have been employed, such as utilizing chemokines to attract T cells and antigen-presenting cells. Chemokine adjuvant strategies may enhance tumor antigen-specific immunity induced by vaccines. Therefore, we utilized chemokine ligand 21 (CCL21) as an adjuvant with a xenogeneic TERT DNA vaccine to induce tumor antigen-specific immunity against TERT-expressing breast cancer. The TERT DNA vaccine consisted of a plasmid containing the COOH terminal end of the TERT (cTERT) gene, encapsulated in multilayered liposomes with hemagglutinating virus of Japan coating. We demonstrated that CCL21 treatment before cTERT DNA vaccine, given intramuscularly, induced significantly higher anti-TERT specific cell-mediated immunity compared to cTERT DNA vaccine alone. Effective tumor antigen-specific immunity was shown both in prophylactic and therapeutic regimens against TS/A murine breast cancer. The study demonstrated that CCL21 administration before cTERT DNA vaccination significantly augmented tumor antigen-specific immunity against breast cancer.

In early-onset Myasthenia Gravis (MG) with anti-acetylcholine receptor antibodies, thymic abnormalities associated with ectopic germinal centers are frequent. miRNAs by acting as post-transcriptional regulators are involved in autoimmunity. To investigate the implication of miRNAs in thymic changes associated with early-onset MG, we performed a miRnome study and data were analyzed with different approaches. miRNAs of interest were further investigated by RT-PCR and transfection experiments for functional tests. First, analyzing specific dysregulated miRNAs, we focused our attention on miR-7-5p and miR-125a-5p, and confirmed by RT-PCR their respective down- and up-regulation in MG thymuses. miR-7 was the most down-regulated thymic miRNA in MG and we observed an inverse correlation between its expression and CCL21 mRNA expression. We next showed that miR-7 down-regulation was due to thymic epithelial cells and by transfecting these cells with miR-7, we demonstrated that it controlled CCL21 release. As CCL21 is essential for germinal center development, we suggested that miR-7 could be involved in thymic changes associated with MG. miR-125a was up-regulated in MG thymuses and is of great interest as it is known to regulate FoxP3 expression, and to modulate the different inflammatory signaling pathways. Thanks to this thymic miRnome study, we also showed the specific dysregulation of miRNA clusters. In particular, we observed that miRNAs localized at the extremity of the X chromosome were down-regulated. This effect seemed linked to their close localization to the fragile X mental retardation 1 gene (FMR1) and the DNA methylation status. Altogether, this miRnome analysis demonstrated that specific thymic miRNAs can be associated with MG and provides novel insights into the pathogenesis of MG.

Chemokines are a superfamily of small cytokines with activities ranging from leukocyte traffick to hematopoiesis, angiogenesis, and tissue organogenesis. Secondary lymphoid-organ chemokine (SLC/CCL21) was originally reported as a chemokine constitutively expressed by stromal cells and high endothelial venules in secondary lymphoid tissues and endothelium of afferent lymphatics, directing CCR7+ cells. More recently, others and we have demonstrated that SLC/CCL21 is up-regulated in different skin inflammatory conditions. Thereafter, this molecule is much more than a constitutive chemokine, which could play a role in effector and regulatory immune functions.

The role of altered levels of insulin, leptin and adiponectin in contributing to the observed increased risk of colon cancer associated with obesity remains to be determined. Elevated insulin and leptin associated with obesity are linked to inflammatory responses. Conversely, adiponectin levels are reduced in obese individuals and this hormone is generally associated with anti-inflammatory responses. Inflammatory cytokines are key components of processes linked with carcinogenesis. Insulin, leptin and adiponectin receptor expression profiles were assessed in human normal, adenomatous polyp and tumour tissue. Insulin, leptin and adiponectin regulation of inflammatory cytokines previously identified as being associated with early events in colon carcinogenesis were further investigated here using a surrogate colon epithelial cell line and a custom designed GeXP assay of the inflammatory cytokines (CCL20, CXCL1, CXCL2, CXCL3, CXCL11, IL1RN, CXCL4, IL8, CCL19, CCL21, CCL23, CCL5, IL10RB and TNFRSF1A). Mean insulin, leptin and adiponectin receptor expression levels were lower in adenomatous polyp samples in comparison with normal and tumour tissue. In contrast to leptin, insulin significantly reduced CCL20 and CXCL11 and increased CXCL3 expression. Full length adiponectin, but not globular adiponectin, induced CCL5, CXCL1, CXCL3 and CCL20 gene expression. GeXP assay permitted measurement of changes in gene expression of cytokines in response to insulin and adiponectin, indicating the potential for insulin and adiponectin regulation of mediators of inflammation associated with early events in colon carcinogenesis.

The oral and esophageal mucosa have been identified as possible sites of HIV/SIV entry following oral infection. Here, gamma/delta (gammadelta) T cells, a multi-functional T cell subset, were assessed at oral/esophageal mucosa and lymphoid sites at the earliest times (1-14 days) post-oral SIV inoculation utilizing quantitative RT-PCR. During these earliest times post-infection, decreased gammadelta TCR mRNA levels were observed at the oral gingiva and esophageal mucosa, while increased levels were observed within regional lymph nodes (cervical and retropharyngeal). Higher lymph node gammadelta TCR levels were associated with increased mRNA expression of the lymphoid homing chemokine/receptor (CCL21/CCR7) pair in these lymph nodes. In contrast to gammadelta TCR levels, CD4 mRNA expression remained relatively stable through 4 days post-infection, and depletion of CD4 T cells was only evident after 7 or 14 days post-infection. The decrease of gammadelta T cell mRNA from mucosal sites and the corresponding increase at lymphoid sites suggest a rapid redistribution of these immune cells at these earliest times post-SIV infection.

Chemokines are important players in the development of allergic contact dermatitis (ACD). The participation of secondary lymphoid tissue chemokine (CCL21) is essential in the induction of the disease due to its expression in lymphatic vessels and in secondary lymphoid organs. Since there is no information about its participation during the effector phase of ACD, we studied this chemokine in patients already diagnosed with ACD, who were challenged with the relevant positive and negative (control) antigens. All patients showed a specific antigen-induced immune response characterized by early expression of inflammatory markers in blood endothelial cells followed by dermal accumulation of mononuclear cells with an important increase in infiltration of CXCR3+ but not of CCR7+ cells. In situ hybridization and immunohistochemistry showed low levels of CCL21 in lymphatic vessels at 2 h, whereas they were significantly increased at 10 and 48 h in all positive patch tests. In contrast, very low expression of this chemokine was observed in skin biopsies from the control site at 48 h. In addition, Langerin+ cells, which were present in dermis from positive patch tests at 2 h, were diminished in number at 10 and 48 h, but a significant number of those cells was still present in dermal areas of the control site at 48 h. We demonstrate for the first time that CCL21, a constitutively expressed chemokine, is strongly upregulated in human lymphatic vessels during a Th1/Tc1 allergic inflammatory response. This can provide the signal required for CCR7+ cells to leave the skin through CCL21-positive lymphatic vessels.

BACKGROUND

Understanding the pathophysiology of chemokine secretion in endometriosis may offer a novel area of therapeutic intervention. This study aimed to identify chemokines differentially expressed in epithelial glands in eutopic endometrium from normal women and those with endometriosis, and to establish the expression profiles of key chemokines in endometriotic lesions.

METHODS

Laser capture microdissection isolated epithelial glands from endometrial eutopic tissue from women with and without endometriosis in the mid-secretory phase of their menstrual cycles. Gene profiling of the excised glands used a human chemokine and receptor cDNA array. Selected chemokines were further examined using real-time PCR and immunohistochemistry.

RESULTS

22 chemokine/receptor genes were upregulated and two downregulated in pooled endometrial epithelium of women with endometriosis compared with controls. CCL16 and CCL21 mRNA was confirmed as elevated in some women with endometriosis compared to controls on individual samples. Immunoreactive CCL16 and CCL21 were predominantly confined to glands in eutopic and ectopic endometrium: leukocytes also stained. Immunoreactive CCL16 was overall higher in glands in ectopic vs. eutopic endometrium from the same woman (P < 0.05). Staining for CCL16 and CCL21 was highly correlated in individual tissues.

CONCLUSIONS

This study provides novel candidate molecules and suggests a potential local role for CCL16 and CCL21 as mediators contributing to the inflammatory events associated with endometriosis.