Type 1 diabetes is characterized by destruction of insulin-producing β cells in the pancreatic islets by effector T cells. Tregs, defined by the markers CD4 and FoxP3, regulate immune responses by suppressing effector T cells and are recruited to sites of action by the chemokine CCL22. Here, we demonstrate that production of CCL22 in islets after intrapancreatic duct injection of double-stranded adeno-associated virus encoding CCL22 recruits endogenous Tregs to the islets and confers long-term protection from autoimmune diabetes in NOD mice. In addition, adenoviral expression of CCL22 in syngeneic islet transplants in diabetic NOD recipients prevented β cell destruction by autoreactive T cells and thereby delayed recurrence of diabetes. CCL22 expression increased the frequency of Tregs, produced higher levels of TGF-β in the CD4+ T cell population near islets, and decreased the frequency of circulating autoreactive CD8+ T cells and CD8+ IFN-γ–producing T cells. The protective effect of CCL22 was abrogated by depletion of Tregs with a CD25-specific antibody. Our results indicate that islet expression of CCL22 recruits Tregs and attenuates autoimmune destruction of β cells. CCL22-mediated recruitment of Tregs to islets may be a novel therapeutic strategy for type 1 diabetes.

BACKGROUND

The FOXP3 mRNA expression and the other regulatory T cell-related molecules were investigated and compared with clinicopathological parameters in human primary breast cancer.

METHODS

This study included 136 breast cancer patients operated in our department from 2003 to 2006. Total RNA was extracted from frozen normal breast and breast cancer tissues, and the expression of FOXP3, IL-10, TGFbeta1 and CCL22 mRNA was evaluated using quantitative real-time RT-PCR.

RESULTS

FOXP3, IL-10, TGFbeta1 and CCL22 mRNA expressions were significantly higher in cancer tissue than in normal tissue, not only at pT1, 2, and 3 stages but also at the DCIS stage. There were positive correlations between FOXP3 and IL-10, FOXP3 and TGFbeta1, as well as FOXP3 and CCL22 mRNA expressions, respectively. FOXP3 and IL-10 mRNA expressions were significantly upregulated in PgR-negative or HER2-positive tumors.

CONCLUSIONS

These results suggest that regulatory T cells are involved in tumor onset and progression in human primary breast cancer, possibly contributing to poor prognosis of patients with breast cancer.

Children that were administered a formalin-inactivated respiratory syncytial virus (FI-RSV) vaccine experienced enhanced respiratory disease, including pulmonary eosinophilia, after contracting a natural RSV infection. RSV vaccine-enhanced disease can be mimicked in BALB/c mice immunized with either FI-RSV or with a recombinant vaccinia virus (vacv) expressing the RSV attachment (G) protein. We have recently demonstrated that memory CD8 T cells directed against the RSV immunodominant M2(82-90) epitope inhibit the development of pulmonary eosinophilia in either vacvG- or FI-RSV-immunized mice by reducing the total number of Th2 cells in the lung after RSV challenge. In this study, we show that memory CD8 T cells specific to a subdominant epitope within the RSV fusion (F) protein fail to inhibit the development of pulmonary eosinophilia after RSV challenge of mice previously co-immunized with vacvF and with either vacvG or FI-RSV. We observed that the inability of RSV F(85)-specific memory CD8 T cells to inhibit the development of pulmonary eosinophilia was largely due to an inadequate total number of F(85)-specific memory CD8 T cells in the lung at early times after RSV challenge. Increasing the number of F(85)-specific memory CD8 T cells after immunization grants these cells the ability to inhibit RSV vaccine-enhanced pulmonary eosinophilia. Moreover, we demonstrate that RSV-specific memory CD8 T cells, when present in sufficient numbers, inhibit the production of the Th2-associated chemokines CCL17 and CCL22. Taken together, these results indicate that RSV-specific memory CD8 T cells may alter the trafficking of Th2 cells and eosinophils into the lung.

Tumour-infiltrating lymphocytes (TILs) favour survival in human colorectal cancer (CRC). Chemotactic factors underlying their recruitment remain undefined. We investigated chemokines attracting T cells into human CRCs, their cellular sources and microenvironmental triggers.

Expression of genes encoding immune cell markers, chemokines and bacterial 16S ribosomal RNA (16SrRNA) was assessed by quantitative reverse transcription-PCR in fresh CRC samples and corresponding tumour-free tissues. Chemokine receptor expression on TILs was evaluated by flow cytometry on cell suspensions from digested tissues. Chemokine production by CRC cells was evaluated in vitro and in vivo, on generation of intraperitoneal or intracecal tumour xenografts in immune-deficient mice. T cell trafficking was assessed on adoptive transfer of human TILs into tumour-bearing mice. Gut flora composition was analysed by 16SrRNA sequencing.

CRC infiltration by distinct T cell subsets was associated with defined chemokine gene signatures, including CCL5, CXCL9 and CXCL10 for cytotoxic T lymphocytes and T-helper (Th)1 cells; CCL17, CCL22 and CXCL12 for Th1 and regulatory T cells; CXCL13 for follicular Th cells; and CCL20 and CCL17 for interleukin (IL)-17-producing Th cells. These chemokines were expressed by tumour cells on exposure to gut bacteria in vitro and in vivo. Their expression was significantly higher in intracecal than in intraperitoneal xenografts and was dramatically reduced by antibiotic treatment of tumour-bearing mice. In clinical samples, abundance of defined bacteria correlated with high chemokine expression, enhanced T cell infiltration and improved survival.

Gut microbiota stimulate chemokine production by CRC cells, thus favouring recruitment of beneficial T cells into tumour tissues.

Cancers subvert the host immune system to facilitate disease progression. These evolved immunosuppressive mechanisms are also implicated in circumventing immunotherapeutic strategies. Emerging data indicate that local tumor-associated DC populations exhibit tolerogenic features by promoting Treg development; however, the mechanisms by which tumors manipulate DC and Treg function in the tumor microenvironment remain unclear. Type III TGF-β receptor (TGFBR3) and its shed extracellular domain (sTGFBR3) regulate TGF-β signaling and maintain epithelial homeostasis, with loss of TGFBR3 expression promoting progression early in breast cancer development. Using murine models of breast cancer and melanoma, we elucidated a tumor immunoevasion mechanism whereby loss of tumor-expressed TGFBR3/sTGFBR3 enhanced TGF-β signaling within locoregional DC populations and upregulated both the immunoregulatory enzyme indoleamine 2,3-dioxygenase (IDO) in plasmacytoid DCs and the CCL22 chemokine in myeloid DCs. Alterations in these DC populations mediated Treg infiltration and the suppression of antitumor immunity. Our findings provide mechanistic support for using TGF-β inhibitors to enhance the efficacy of tumor immunotherapy, indicate that sTGFBR3 levels could serve as a predictive immunotherapy biomarker, and expand the mechanisms by which TGFBR3 suppresses cancer progression to include effects on the tumor immune microenvironment.

Type-1 and type-2 lung granulomas, respectively, elicited by bead immobilized Mycobacteria bovis and Schistosoma mansoni egg antigens (Ags) display different patterns of chemokine expression. This study tested the hypothesis that chemokine expression patterns were related to upstream cytokine signaling. Using quantitative transcript analysis, we defined expression profiles for 16 chemokines and then examined the in vivo effects of neutralizing antibodies against interferon-gamma (IFN-gamma), interleukin (IL)-4, IL-10, IL-12, and IL-13. Transcripts for CXCL2, -5, -9, -10, and -11 and the CCL chemokine, CCL3, and lymphotactin (XCL1), were largely enhanced by Th1-related cytokines, IFN-gamma or IL-12. Transcripts for CCL11, CCL22, CCL17, and CCL1 were enhanced largely by Th2-related cytokines, IL-4, IL-10, or IL-13. Transcripts for CCL4, CCL2, CCL8, CCL7, and CCL12 were potentially induced by either Th1- or Th2-related cytokines, although some of these showed biased expression. IFN-gamma and IL-4 enhanced the greatest complement of transcripts, and their neutralization had the greatest anti-inflammatory effect on type-1 and type-2 granulomas, respectively. Th1/Th2 cross-regulation was evident because endogenous Th2 cytokines inhibited type-1, whereas Th1 cytokines inhibited type-2 biased chemokines. These findings reveal a complex cytokine-chemokine regulatory network that dictates profiles of local chemokine expression during T cell-mediated granuloma formation.

Chemokines and their receptors play a pivotal role in controlling T cell trafficking in immunity and inflammation. Two chemokines, CCL17 and CCL22, activate the chemokine receptor CCR4, expressed on functionally distinct subsets of T cells: cutaneous leukocyte-associated antigen (CLA)+ skin-homing, T helper (Th) 2, and CD25+ T suppressor cells. Here, we compared the ability of CCL17 and CCL22 to promote CCR4 internalization as a mechanism of regulation of receptor function on human Th2 cells. We report that CCL22 is a potent and rapid inducer of CCR4 internalization, while CCL17 is not. CCR4 internalization does not require G protein coupling, while being dependent on lipid rafts integrity and clathrin-coated pits functionality. Cell surface disappearance of CCR4 is rapidly reversed upon removal of exogenous ligand by virtue of receptor recycling. CCR4 internalization leads to a loss of functional responsiveness, while recovery of surface expression leads to re-acquisition of chemotactic sensitivity of Th2 cells. The differential CCR4 desensitization and internalization reported here and the distinct expression patterns of CCL17 and CCL22 observed in vivo suggest that while CCL17 may act first on CCR4 at the endothelial surface to promote vascular recognition, CCL22 could subsequently engage the receptor within the tissue microenvironment to guide cellular localization.

Hodgkin lymphoma (HL) is characterized by a minority of neoplastic Hodgkin-Reed Sternberg (HRS) cells surrounded by a non-neoplastic reactive infiltrate. As immunological mechanisms appear to be crucial in classical HL pathogenesis, altered serum chemokine levels might be related to disease activity. Serum levels of nine chemokines were examined in 163 untreated HL patients and 334 controls. We investigated single nucleotide polymorphisms (SNPs) for association with serum CCL17 (thymus and activation-regulated chemokine, TARC) levels and HL susceptibility. Serum CCL17 and CCL22 (macrophage-derived chemokine, MDC) levels were significantly increased in 82% and 57% of the HL patients. Nodular sclerosis cases showed increased serum CCL17 and CCL22 levels (P < 0.001) and serum levels were correlated with Ann Arbor stage. Of nine patients with pre- and post-treatment serum samples, the majority showed decreased CCL17 and CCL22 levels after treatment. HRS cells expressed CCL17 and CCL22 in 77% and 75% of 74 cases. Three SNPs showed a trend of increased serum CCL17 levels with minor alleles in controls, but were not associated with HL susceptibility. CCL17 and CCL22 were the only chemokines with increased serum levels in the vast majority of HL patients, which provides further insight into the molecular mechanism(s) leading to infiltrations of reactive lymphocytes in HL.

BACKGROUND

Dendritic cells (DCs) are often produced by granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) stimulation of monocytes. To improve the effectiveness of DC adoptive immune cancer therapy, many different agents have been used to mature DCs. We analyzed the kinetics of DC maturation by lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) induction in order to characterize the usefulness of mature DCs (mDCs) for immune therapy and to identify biomarkers for assessing the quality of mDCs.

METHODS

Peripheral blood mononuclear cells were collected from 6 healthy subjects by apheresis, monocytes were isolated by elutriation, and immature DCs (iDCs) were produced by 3 days of culture with GM-CSF and IL-4. The iDCs were sampled after 4, 8 and 24 hours in culture with LPS and IFN-gamma and were then assessed by flow cytometry, ELISA, and global gene and microRNA (miRNA) expression analysis.

RESULTS

After 24 hours of LPS and IFN-gamma stimulation, DC surface expression of CD80, CD83, CD86, and HLA Class II antigens were up-regulated. Th1 attractant genes such as CXCL9, CXCL10, CXCL11 and CCL5 were up-regulated during maturation but not Treg attractants such as CCL22 and CXCL12. The expression of classical mDC biomarker genes CD83, CCR7, CCL5, CCL8, SOD2, MT2A, OASL, GBP1 and HES4 were up-regulated throughout maturation while MTIB, MTIE, MTIG, MTIH, GADD45A and LAMP3 were only up-regulated late in maturation. The expression of miR-155 was up-regulated 8-fold in mDCs.

CONCLUSIONS

DCs, matured with LPS and IFN-gamma, were characterized by increased levels of Th1 attractants as opposed to Treg attractants and may be particularly effective for adoptive immune cancer therapy.

Tumor-induced immune suppression involves the accumulation of immune-suppressive infiltrates in the microenvironment. This study demonstrates increased numbers of CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs) in the lungs of C57BL/6 mice bearing a metastatic Lewis lung carcinoma (LLC) variant. These Tregs suppressed the proliferation of endogenous CD4(+)CD25(-) cells and expressed higher levels of the chemokine receptor CCR4 than other types of T cells. LLC-bearing lungs secreted elevated levels of the CCR4-associated chemokine CCL22 compared with normal lungs. However, CCL22 was not secreted by LLC or normal epithelial controls, suggesting that CCL22 is secreted by a nonepithelial component of the microenvironment. Migration assays revealed that medium conditioned by LLC-bearing lungs selectively recruited Tregs at higher frequencies than did medium conditioned by normal lungs. Neutralization of CCL22 significantly reduced this selective recruitment toward both conditioned media. A series of immunomagnetic isolations, FACS, and flow cytometric analyses were used to isolate different cellular fractions from both normal and LLC-bearing lungs. When isolated, only the NK-containing fractions secreted CCL22, and the same fraction isolated from LLC-bearing lungs secreted higher levels. Depletion of NK cells from both normal and LLC-bearing lung tissue significantly reduced CCL22 secretion, suggesting that a large portion of secreted CCL22 is NK cell dependent. Flow cytometric analysis of the lung NK compartments revealed no significant increase in NK cell numbers across LLC-bearing lung tissue as a whole as compared with normal tissue. However, immunofluorescent staining revealed an increased frequency of NK cells at the tumor periphery that were closely associated with the elevated FoxP3(+) infiltrate.

Chemokines and their corresponding receptors are crucial for the recruitment of lymphocytes into the lymphoid organs and for its organization acting in a multistep process. Tissues affected by autoimmune disease often contain ectopic lymphoid follicles which, in the case of autoimmune thyroid disorders, are highly active and specific for thyroid Ags although its pathogenic role remains unclear. To understand the genesis of these lymphoid follicles, the expression of relevant cytokines and chemokines was assessed by real time PCR, immunohistochemistry and by in vitro assays in autoimmune and nonautoimmune thyroid glands. Lymphotoxin alpha, lymphotoxin beta, C-C chemokine ligand (CCL) 21, CXC chemokine ligand (CXCL) 12, CXCL13, and CCL22 were increased in thyroids from autoimmune patients, whereas CXCL12, CXCL13, and CCL22 levels were significantly higher in autoimmune glands with ectopic secondary lymphoid follicles than in those without follicles. Interestingly, thyroid epithelium produced CXCL12 in response to proinflammatory cytokines providing a possible clue for the understanding of how tissue stress may lead to ectopic follicle formation. The finding of a correlation between chemokines and thyroid autoantibodies further suggests that intrathyroidal germinal centers play a significant role in the autoimmune response. Unexpectedly, the percentage of circulating CXCR4(+) T cells and CCR7(+) B and T cells (but not of CXCR5) was significantly reduced in PBMCs of patients with autoimmune thyroid disease when they were compared with their intrathyroidal lymphocytes. This systemic effect of active intrathyroidal lymphoid tissue emerges as a possible new marker of thyroid autoimmune disease activity.

The combination of galunisertib, a transforming growth factor (TGF)-β receptor (R)1 kinase inhibitor, and lomustine was found to have antitumor activity in murine models of glioblastoma.

Galunisertib (300 mg/day) was given orally 14 days on/14 days off (intermittent dosing). Lomustine was given as approved. Patients were randomized in a 2:1:1 ratio to galunisertib + lomustine, galunisertib monotherapy, or placebo + lomustine. The primary objective was overall survival (OS); secondary objectives were safety, pharmacokinetics (PKs), and antitumor activity.

One hundred fifty-eight patients were randomized: galunisertib + lomustine (N = 79), galunisertib (N = 39), and placebo + lomustine (N = 40). Baseline characteristics were: male (64.6%), white (75.3%), median age 58 years, ECOG performance status (PS) 1 (63.3%), and primary glioblastoma (93.7%). The PKs of galunisertib were not altered with lomustine, and galunisertib had a median half-life of ∼8 hours. Median OS in months (95% credible interval [CrI]) for galunisertib + lomustine was 6.7 (range: 5.3-8.5), 8.0 (range: 5.7-11.7) for galunisertib alone, and 7.5 (range: 5.6-10.3) for placebo + lomustine. There was no difference in OS for patients treated with galunisertib + lomustine compared with placebo + lomustine [P (HR < 1) = 26%]. Median progression-free survival of ∼2 months was observed in all 3 arms. Among 8 patients with IDH1 mutation, 7 patients were treated with galunisertib (monotherapy or with lomustine); OS ranged from 4 to 17 months. Patients treated with galunisertib alone had fewer drug-related grade 3/4 adverse events (n = 34) compared with lomustine-treated patients (10% vs 26%). Baseline PS, post-discontinuation of bevacizumab, tumor size, and baseline levels of MDC/CCL22 were correlated with OS.

Galunisertib + lomustine failed to demonstrate improved OS relative to placebo + lomustine. Efficacy outcomes were similar in all 3 arms.

NCT01582269, ClinicalTrials.gov.

The pathogenesis of rheumatoid arthritis (RA) and psoriatic arthritis (PsA) involves an abnormal chemokine regulation. The chemokine receptor CCR4 is necessary for T cell migration to the skin. We, therefore, studied if CCR4 and its ligand macrophage-derived chemokine (MDC/CCL22) could participate in spreading the disease between skin and joints by examining RA, PsA and osteoarthritis (OA) patients. In synovial fluid from RA and PsA patients we observed a significantly higher MDC/CCL22 level compared to OA patients. Additionally, the MDC/CCL22 protein was found to be elevated in RA and PsA plasma compared to OA and healthy volunteers. Flow cytometry revealed that most CD4(+)CCR4(+) lymphocytes also co-expressed CD45RO. Neither the MDC/CCL22 level nor the expression of CCR4 correlated to CRP. Immunohistochemistry of the RA and OA synovial membrane demonstrated CCR4 to be expressed by mononuclear cells and endothelial cells. Our results show that MDC/CCL22 is present within the synovial membrane of RA and OA patients and in high amount in the synovial fluid of patients with RA and PsA. This will enable migration of CCR4 expressing memory cells supporting that MDC/CCR4 could play a role in attracting skin specific memory T cells to the joints.

OBJECTIVE

In contrast to many other malignancies, in uveal melanoma (UM) the presence of an immune infiltrate is associated with a bad prognosis. An analysis of the different functional phenotypes of tumor-infiltrating leukocytes (TIL) and a comparison with the genetic background of the tumors may help to explain this apparent anomaly.

METHODS

We performed a comprehensive immunohistochemical study by evaluating the density of CD8(+) and CD4(+) T lymphocytes, forkhead box p3 (Foxp3(+)) regulatory T cells (Tregs), and CD68(+) and CD68(+)CD163(+) macrophages in 43 cases of UM in relation to tumor characteristics. Expression of the chemokines CCL2, CCL17, and CCL22 in cultured human UM cells and peripheral blood monocytes was analyzed by quantitative PCR (qPCR).

RESULTS

The presence of TILs was highly variable between tumors and was dominated by CD8(+) T cells with fewer CD4(+) T cells and Tregs. When tumors were infiltrated by immune cells, the infiltrate generally comprised all different subsets of lymphocytes (P < 0.001) and M2 macrophages (P < 0.001). Different T-cell ratios did not influence clinical outcome. In addition, the presence of TIL correlated with the loss of one chromosome 3 (P < 0.04). UM cells express CCL2 and CCL22, two chemokines known to mediate trafficking of immune cells to the tumor.

CONCLUSIONS

All studied subtypes of tumor-infiltrating immune cells were collectively increased and showed an association with monosomy of chromosome 3 suggesting that tumor intrinsic factors control the leukocyte influx, possibly through local chemokine secretion.

Basal cell carcinoma (BCC), the most common human cancer, undergoes spontaneous regression in certain circumstances, which is potentially immune-mediated. To understand the immune response surrounding BCCs, we characterized the genomic, protein, and cellular microenvironment associated with BCC in comparison to normal skin. Our results demonstrated the following: (1) CD4+ CD25+ Foxp3+ surround epithelial tumor aggregates; (2) Immature dendritic cells (DCs) were abundant in the tumor microenvironment; (3) BCC showed increased expression of IL-4, IL-10, and CCL22 and increased expression of interferon-associated genes (IFI27, IRF1, IRF7, and G1P2) and IL-12/23, gene indicating a Th2 dominant microenvironment. Our findings suggest a dynamic state within the immune microenvironment associated with BCC. The finding of phenotypic T regs, in conjunction with immature DCs and Th2 cytokines, suggests an attenuated state of immunity to human BCC. In contrast, abundant CD8+ T cells, an interferon signal, and IL-12/23 suggest partial host antitumor response. A better understanding of these opposing forces within the immune microenvironment may facilitate development of more potent immune-based treatment for BCC and other human carcinomas.

We previously reported that chronic stimulation with low, noncytotoxic doses of extracellular adenosine triphosphate (ATP) induced a distorted maturation of dendritic cells (DCs) and impaired their capacity to initiate T-helper (Th) 1 responses in vitro. Here, we examined the effects of ATP on chemokine-receptor expression and chemokine production by DCs. ATP strongly induced expression of CXC chemokine receptor 4 on both immature and lipopolysaccharide (LPS)-stimulated DCs and slightly up-regulated CC chemokine receptor (CCR) 7 on both DC types. In contrast, ATP reduced CCR5 expression on immature DCs. These effects were confirmed at both the messenger RNA and protein levels and were not produced by uridine triphosphate (UTP). Consistent with the changed receptor expression, ATP increased migration and intracellular calcium of immature and mature DCs to stromal-derived factor 1 (CXC ligand [CXCL] 12) and macrophage inflammatory protein [MIP] 3 beta (CC ligand [CCL] 19), whereas responses to MIP-1 beta (CCL4) were reduced. DCs are an important source of chemokines influencing recruitment of distinct T-lymphocyte subsets. ATP, but not UTP, significantly reduced LPS-induced production of interferon-inducible protein 10 (CXCL10) and regulated upon activation, normal T-cell expressed and secreted chemokine (CCL5); increased secretion of macrophage-derived chemokine (CCL22); and did not change production of thymus and activation-regulated chemokine (CCL17). Consistent with these findings, supernatants from ATP-treated mature DCs attracted Th1 and T-cytotoxic 1 cells less efficiently, whereas migration of Th2 and T cytotoxic 2 cells was not affected. Our data suggest that ATP provides a signal for enhanced lymph node localization of DCs but that it may, at the same time, diminish the capacity of DCs to amplify type 1 immune responses.

Thymus and activation-regulated chemokine (TARC)/CCL17 and macrophage-derived chemokine (MDC)/CCL22 are a pair of CC chemokines known to selectively attract T(h)2 type memory T cells via CCR4. Here we examined circulating levels of TARC and MDC in patients with atopic dermatitis (AD) and control subjects by using plasma samples, which reflect blood contents of chemokines more accurately than serum samples. The plasma levels of TARC and MDC were significantly elevated in AD patients. These values also strongly correlated with disease severity and serum lactate dehydrogenase levels, and weakly correlated with serum total IgE levels and blood eosinophilia. Previous studies demonstrated TARC immunoreactivity in the epidermal layer of AD lesional skin and production of TARC by a human keratinocytic cell line HaCaT upon stimulation with IFN-gamma. Here we demonstrated MDC immunoreactivity in the epidermal layer of AD skin at levels stronger than that of TARC. Furthermore, primary epidermal keratinocytes expressed both TARC and MDC mRNA upon stimulation with IFN-gamma, but efficiently secreted only MDC. These results suggest a post-transcriptional regulation in TARC production. IFN-gamma also induced TARC and MDC mRNA in mouse skin. Collectively, both TARC and MDC play important roles in the local accumulation of T(h)2 cells in AD lesional skin. Production of T(h)2-attracting chemokines by epidermal keratinocytes upon treatment with IFN-gamma, which is also the potent inducer of T(h)1-attracting chemokines, may underline the pivotal role of IFN-gamma in the chronic phase of AD where both T(h)1 and T(h)2 responses are mixed.

Atopic dermatitis (AD) is considered to be Th2 cell-mediated disorder. In most infants with AD, AD may be induced by food allergy. In the early stage of infantile AD, it is unclear whether there are changes in serum Th2 chemokines or in Th2 chemokine production by peripheral blood mononuclear cells (PBMC). Thirty-four patients with AD were examined (mean age, 4.5 months; female:male, 18:16). Ten age-matched infants with no history of allergic disease were used as controls. Thirty of these 34 patients were sensitized with ovalbumin (OVA; radioallergrosolvent score of >2). Serum levels of CCL17, CCL22, and CCL27 were measured with enzyme-linked immunosolvent assay (ELISA) kits and their correlation with the severity of skin lesions, defined by the scoring atopic dermatitis (SCORAD) index, was analyzed. The amounts of TNF-alpha, CCL17, CCL22, and CCL27 in the culture supernatants of PBMC from OVA-sensitized AD infants after stimulation with OVA were estimated with ELISA kits. Elevated serum CCL17, CCL22, and CCL27 levels significantly correlated with SCORAD index (r = 0.7181, p < 0.001; r = 0.5354, p < 0.005; r = 0.8312, p < 0.0001, respectively). CCL22 levels produced by PBMC from OVA-sensitized infants with AD reflected serum CCL22 levels. Only six of 30 OVA-sensitized patients in whom the skin signs increased immediately after OVA intake showed markedly high titers of TNF-alpha produced by PBMC after stimulation with OVA. These high TNF-alpha titers correlated significantly with serum CCL27 levels (r = 0.7181, p < 0.001). Serum concentrations of CCL17, CCL22, and CCL27 correlate well with the extent and intensity of AD in infants. Of the three Th2 chemokines examined, serum CCL27 correlated most significantly with the severity of AD. Thus, the peripheral immune responses of infantile AD patients are skewed to a Th2 dominant bias.

Mycoplasma pneumoniae causes acute and chronic lung infections in humans, leading to a variety of pulmonary and extrapulmonary sequelae. Of the airway complications of M. pneumoniae infection, M. pneumoniae-associated exacerbation of asthma and pediatric wheezing are emerging as significant sources of human morbidity. However, M. pneumoniae products capable of promoting allergic inflammation are unknown. Recently, we reported that M. pneumoniae produces an ADP-ribosylating and vacuolating toxin termed the community-acquired respiratory distress syndrome (CARDS) toxin. Here we report that naive mice exposed to a single dose of recombinant CARDS (rCARDS) toxin respond with a robust inflammatory response consistent with allergic disease. rCARDS toxin induced 30-fold increased expression of the Th-2 cytokines IL-4 and IL-13 and 70- to 80-fold increased expression of the Th-2 chemokines CCL17 and CCL22, corresponding to a mixed cellular inflammatory response comprised of a robust eosinophilia, accumulation of T cells and B cells, and mucus metaplasia. The inflammatory responses correlate temporally with toxin-dependent increases in airway hyperreactivity characterized by increases in airway restriction and decreases in lung compliance. Furthermore, CARDS toxin-mediated changes in lung function and histopathology are dependent on CD4(+) T cells. Altogether, the data suggest that rCARDS toxin is capable of inducing allergic-type inflammation in naive animals and may represent a causal factor in M. pneumoniae-associated asthma.

Helicobacter pylori infects the stomach and duodenal mucosa. T cells are important components of the H. pylori-induced immune response, but little is currently known about how these cells are recruited to the infected mucosa. Here, we have characterized stomach and duodenal T cells isolated from H. pylori-infected and noninfected subjects with regard to subtype, expression of homing and chemokine receptors, and in vitro reactivity to H. pylori antigens. Higher numbers of CD4(+) but similar numbers of CD8(+) lamina propria T cells were isolated from stomach biopsies from H. pylori-positive compared to H. pylori-negative individuals. CD4(+) T cells from infected stomach expressed increased levels of the homing receptor L-selectin and the chemokine receptor CCR4 compared to CD4(+) T cells from uninfected stomach. Infected stomach mucosa also contained increased levels of the CCR4 chemokine ligand MDC/CCL22. In contrast, comparable numbers of CD4(+) T cells with similar receptor expression were isolated from the duodenum of H. pylori-positive and H. pylori-negative individuals. In vitro proliferation of mucosal T cells was strongly enhanced by the addition of interleukin-2 (IL-2) and IL-7 to the cell cultures. Using this approach, H. pylori-specific T-cell responses were detected in stomach CD4(+) T cells from H. pylori-positive but not H. pylori-negative individuals. Duodenal T cells from only a few individuals responded to H. pylori stimulation, and the responsiveness was not restricted to H. pylori-positive individuals, suggesting limited H. pylori specificity in the duodenum and possible cross-reactivity with antigens from other bacteria in this compartment. In conclusion, these results suggest that H. pylori-specific CD4(+) T cells preferentially home to and accumulate in the infected stomach and that L-selectin and CCR4/MDC are important for this recruitment.

BACKGROUND

In animal models administration of immunostimulatory DNA sequences preferentially elicits T(H)1-dominated (type 1-dominated) immunity and can inhibit developing or ongoing T(H)2 (type 2) responses.

OBJECTIVE

Our objective was to investigate this phenomenon in humans.

METHODS

In a randomized, third party-blinded, placebo-controlled, proof-of-concept study conducted entirely in the winter in 19 adults with ragweed allergy, we administered 6 subcutaneous injections of purified Amb a 1 linked to the 22-base-long immunostimulatory phosphorothioate oligodeoxyribonucleotide 1018 (Amb a 1-immunostimulatory DNA sequence conjugate [AIC]). Before the course of AIC or placebo injections and 2 and 16 weeks afterward, we measured recall responses to ragweed, streptokinase, and PHA in short-term primary culture of fresh PBMCs after restimulation with antigen. We quantified regulatory cytokine and chemokine responses characteristic of T(H)2 immunity (IL-5, IL-13, CCL17 [TARC], and CCL22 [MDC]), and T(H)1 immunity (IFN-gamma, CXCL9 [Mig], and CXCL10 [IP-10]), as well as IL-10, a cytokine sometimes linked to regulatory T-cell populations.

RESULTS

We demonstrated for the first time that human systemic in vivo ragweed-specific T(H)2 responses were selectively redirected toward T(H)1 responses, with significant increases in IFN-gamma, CXCL9, and CXCL10 and significant decreases in IL-5, CCL17, and CCL22 found at 2 and 16 weeks after the sixth injection. Cytokine and chemokine responses to the unrelated bacterial antigen streptokinase and the global capacity to mount immune responses on polyclonal activation with PHA did not change. No clinically significant systemic or local allergic reactions were associated with AIC or placebo injections.

CONCLUSIONS

AIC, injected in concentrations that were approximately 40-fold lower than those used in most murine studies published to date, led to a prolonged shift from T(H)2 immunity toward T(H)1 immunity and appeared to be safe. This novel approach has the potential for immune redirection in human immediate hypersensitivity diseases.

Chemokines are important mediators of macrophage and T-cell recruitment in a number of inflammatory pathologies, and chemokines expressed in atherosclerotic lesions may play an important role in mononuclear cell recruitment and macrophage differentiation. We have analyzed the expression of the linked chromosome 16q13 genes that encode macrophage-derived chemokine (MDC/CCL22), thymus- and activation-regulated chemokine (TARC/CCL17), and the CX(3)C chemokine fractalkine (CX(3)CL1) in primary macrophages and human atherosclerotic lesions by reverse transcription-polymerase chain reaction and immunohistochemistry. We show that macrophage expression of the chemokines MDC, fractalkine, and TARC is upregulated by treatment with the Th2-type cytokines interleukin-4 and interleukin-13. High levels of MDC, TARC, and fractalkine mRNA expression are seen in some, but not all, human arteries with advanced atherosclerotic lesions. Immunohistochemistry shows that MDC, fractalkine, and TARC are expressed by a subset of macrophages within regions of plaques that contain plaque microvessels. We conclude that MDC, fractalkine, and TARC, which are chromosome 16q13 chemokines, could play a role in mononuclear cell recruitment into atherosclerotic lesions and influence the subsequent inflammatory response. Macrophage-expressed chemokines upregulated by interleukin-4 may be useful surrogate markers for the presence of Th2-type immune responses in human atherosclerotic lesions.

In human vitiligo, cutaneous depigmentation involves cytotoxic activity of autoreactive T cells. It was hypothesized that depigmentation can progress in the absence of regulatory T cells (Treg). The percentage of Treg among skin infiltrating T cells was evaluated by immunoenzymatic double staining for CD3 and FoxP3, revealing drastically reduced numbers of Treg in non-lesional, perilesional and lesional vitiligo skin. Assessment of the circulating Treg pool by FACS analysis of CD4, CD25, CD127 and FoxP3 expression, and mixed lymphocyte reactions in presence and absence of sorted Treg revealed no systemic drop in the abundance or activity of Treg in vitiligo patients. Expression of skin homing receptors CCR4, CCR5, CCR8 and CLA was comparable among circulating vitiligo and control Treg. Treg from either source were equally capable of migrating towards CCR4 ligand and skin homing chemokine CCL22, yet significantly reduced expression of CCL22 in vitiligo skin observed by immunohistochemistry may explain failure of circulating, functional Treg to home to the skin in vitiligo. The paucity of Treg in vitiligo skin is likely crucial for perpetual anti-melanocyte reactivity in progressive disease.

Macrophages are key effectors in demyelinating diseases of the central and peripheral nervous system by phagocytosing myelin and releasing immunoregulatory mediators. Here, we report on a distinct, a priori anti-inflammatory reaction of macrophages phagocytosing myelin upon contact with damaged nerve tissue. Macrophages rapidly invaded peripheral (sciatic) and central (optic) nerve tissues in vitro, readily incorporated myelin and expressed high levels of phagocytosis-associated molecules (e.g., Fc and scavenger receptors). In contrast, factors involved in antigen presentation (MHC class-II, CD80, CD86) revealed only a restricted expression. In parallel, a highly ordered appearance of cytokines and chemokines was detected. IL-10, IL-6, CCL22, and CXCL1 were immediately but transiently induced, whereas CCL2, CCL11, and TGFbeta revealed more persisting levels. Such a profile would attract neutrophils, monocytes/macrophages, and Th2 cells as well as bias for a Th2-supporting environment. Importantly, proinflammatory/Th1-supporting factors, such as TNFalpha, IL-12p70, CCL3, and CCL5, were not induced. Still the simultaneous presence of TGFbeta and IL-6 could assist Th17 development, further depending on yet not present IL-23. The release pattern was clearly distinct from reactive phenotypes induced in isolated macrophages and microglia upon treatment with IL-4, IL-13, bacterial lipopolysaccharide, IFNgamma, or purified myelin. Nerve-exposed macrophages thus commit to a unique functional orientation.