Impact of regulatory T cell therapy on immune cell composition and fetal survival rate in abortion prone mice.

CONTEXT: Implantation of fertilised eggs and survival of a semi-allogenic embryo rely on the interactions between the cells and molecules preparing the uterus. We investigated the effect of regulatory T cell (Treg) therapy on the mechanism of local immune tolerance of mice prone to spontaneous abortion. METHODS: Naive T cells were stimulated in vitro with 17ß-oestradiol (E2), progesterone (P4) and TGF-ß1 for 96h to generate induced Tregs (iTreg). The iTregs were injected into DBA/2-mated pregnant CBA/J female mice (abortion prone model). On day 14 of pregnancy, mice were killed and decidual and placental tissues were collected for cellular composition analysis. RESULTS: Abortion prone mice (PBS treated) showed significantly lower survival rates (P <0.0001), increased CD3+ CD8+ (P <0.05), lower IDO+ (P <0.05) and increased natural killer cells (uNK) cell numbers (P <0.001) in the uterus, as well increased NK cells in the placenta (P <0.05) than in normal pregnant mice (CBA/J×BALB/c). Adoptive transfer of iTregs increased fetal survival in abortion-prone mice (P <0.01) and histopathological evaluation revealed a significantly decreased number of uNK cells in the uterus of TGF-ß1-, E2- and P4-iTregs (P<0.05, P<0.0001 and P<0.05, respectively) than in the PBS treated group. In the placenta, we found significantly lower numbers of uNK cells from TGF-ß1-, E2- and P4-iTregs than in the PBS treated group (P <0.05, P <0.05 and P <0.01, respectively). CONCLUSIONS: We propose that modulation of uterine NK cell activity through immunotherapy using Treg cells should be given more attention as an immunological strategy in the treatment of recurrent miscarriage.

Adoptive cell therapy with induced regulatory T cells normalises the abortion rate in abortion-prone mice.

Ovarian hormones drive invivo generation of regulatory T cells (Tregs) during pregnancy. Little is known about the therapeutic potential of invitro hormone-derived Tregs in pregnancy loss. We investigated the effects of hormone-induced Tregs in a murine model of abortion. CD4+CD25- T cells were isolated from the spleens of CBA/J mice and stimulated with either 17ß-oestradiol (E2), progesterone (P4) or transforming growth factor-ß1 (TGFB1) plus retinoic acid (RA) for 4 days to generate induced Tregs (iTregs). On Days 1-4 of gestation, DBA/2-mated pregnant CBA/J female mice (abortion prone) were injected intravenously with iTregs or Tregs isolated from normal BALB/c-mated pregnant CBA/J mice (np-Tregs). On Day 14, the number of resorbed fetuses was assessed. Serum interferon (IFN)-γ and uterine forkhead box p3 (Foxp3) expression was analysed by ELISA and immunohistochemistry respectively. Using a 3H-thymidine incorporation assay, isolated CD4+CD25+ Tregs induced by the different treatments suppressed the proliferation of CD4+CD25- T cells. Adoptive transfer of iTregs (from all induction groups) significantly decreased fetal resorption in abortion-prone mice. There were no significant changes in serum IFN-γ concentrations after the adoptive transfer of iTregs or np-Tregs. Immunohistochemistry revealed significantly higher Foxp3 expression in gravid uteri from mice injected with np-Tregs and P4-induced iTregs than in the phosphate-buffered saline-treated group. The findings of this study indicate a potential therapeutic benefit of invitro-induced Tregs in patients with recurrent abortion.

Major histocompatibility complex class II transactivator gene polymorphism: associations with Löfgren's syndrome.

The major histocompatibility complex (MHC) class II transactivator (MHC2TA) is known as a master regulator for expression of MHC class II molecules. In the present study, we investigated the influence on the risk for sarcoidosis of two variants of the MHC2TA gene, selected from previous association studies of inflammatory diseases. Seven hundred and twenty-eight sarcoidosis patients and 873 controls matched by ethnicity were included in the study. Patients were classified as with Löfgren's syndrome (or not) as subphenotypes. Individuals were genotyped for two single nucleotide polymorphisms (SNPs) of the MHC2TA gene, rs3087456 A/G and rs11074932 C/T, and were human leukocyte antigen (HLA)-DRB1-typed. After correction for multiple testing, our data showed a significant association with Löfgren's syndrome in allelic model for the rs3087456 SNP, which was not detected in non-Löfgren's patients. A similar trend was noted for the rs11074932 SNP. These risk factors were independent of HLA-DRB1*03, which is known to be associated with Löfgren's syndrome. The finding of a new genetic association between Löfgren's syndrome and MHC2TA gene polymorphisms, which seems independent of HLA-DRB1*03 and relates to the expression of MHC class II molecules, strongly supports the idea that Löfgren's syndrome is a separate disease entity.

Increased levels of nerve growth factor in the airways of patients with sarcoidosis.

OBJECTIVES: Nerve growth factor (NGF) is a potent neuronal growth factor with inflammatory properties that recently has been proposed to be of importance in airway pathology. A role for NGF in the inflammatory granulomatous lung disease sarcoidosis is not well elucidated. The aims of this study were to investigate the secreted levels of NGF in bronchoalveolar lavage fluid (BALF) from sarcoidosis patients compared with patients with resolved disease, patients with another granulomatous disease--chronic beryllium disease (CBD)--and healthy subjects and also to investigate the relationship between NGF levels and markers of inflammation. METHODS AND RESULTS: NGF levels in BALF from 56 patients with active sarcoidosis (22 with Löfgren's syndrome), nine subjects with resolved sarcoidosis, six patients with CBD, and 31 healthy subjects were compared. A 10-fold elevation of NGF levels was found in patients with active sarcoidosis compared with subjects with clinically resolved sarcoidosis, patients with CBD and healthy subjects. In sarcoidosis patients, positive correlations between concentrations of NGF and lymphocytes, eosinophils and interferon-gamma, interleukin (IL)-4, IL-10, IL-12 were found. CONCLUSIONS: We demonstrate that secreted levels of NGF are markedly enhanced in the airways in active pulmonary sarcoidosis. Furthermore, a relationship between NGF and pulmonary inflammation in sarcoidosis is supported.

Analysis of regulatory T cell associated forkhead box P3 expression in the lungs of patients with sarcoidosis.

In pulmonary sarcoidosis, the typical T helper 1-mediated immune response in the lungs has been proposed to be co-ordinated by regulatory T cells; however, their exact role needs to be clarified. We used real-time polymerase chain reaction to study genes involved in regulatory T cell functions in CD4+ T cells isolated from bronchoalveolar lavage fluid (BALF) of patients (n = 24) and healthy subjects (n = 7). The genes included the transcription factor forkhead box P3 (FoxP3), interleukin (IL)-10, transforming growth factor-beta1 and chemokine receptor 2 (CCR2). The same genes were also studied in isolated BALF CD4+ T cell receptor AV2S3+ and AV2S3(-) T cells of patients with lung-restricted AV2S3 T cell expansions (n = 12). Intracellular staining of the FoxP3 protein was performed additionally in 14 patients and nine healthy subjects. mRNA expression of FoxP3, CCR2 and IL-10 was decreased significantly in BALF CD4+ T cells of patients. Flow cytometric analysis of CD4+ T cells also demonstrated a decreased frequency of FoxP3+ cells in the BALF and blood of sarcoidosis patients as well as a reduced intensity (mean fluorescence intensity) of FoxP3 expression in BALF FoxP3+ cells of patients. BALF CD4+AV2S3+ T cells expressed significantly lower levels of FoxP3 and CCR2 mRNA versus BALF CD4+AV2S3- T cells. The main conclusion of our study is that there is a reduced expression of regulatory T cell associated genes in BALF CD4+ T cells in sarcoidosis. In addition, our data suggest an effector function of AV2S3+ lung-accumulated T cells in sarcoidosis.

Reduced Th1 response in the lungs of HLA-DRB1*0301 patients with pulmonary sarcoidosis.

To investigate why human leukocyte-associated antigen-DRB1*0301 (HLA-DRB1*0301) positive Scandinavian patients have a better prognosis than HLA-DRB1*0301 negative patients, the present authors examined patterns of cytokine expression in bronchoalveolar lavage (BAL) cells and BAL fluid (BALF) from patients with pulmonary sarcoidosis and controls. Using real-time PCR, the mRNA expression of selected cytokines in BAL cells from newly diagnosed, untreated nonsmoking patients (n=25) and controls (n=11) was quantified. Cytokine protein levels in BALF from patients (n=34) and controls (n=11) were assessed using cytometric bead array. The patients were evaluated and stratified into two subgroups: HLA-DRB1*0301 positive (all with an acute onset) and HLA-DRB1*0301 negative (all with an insidious onset). When comparing patients and controls, BAL cells of the patients expressed significantly higher levels of interferon (IFN)-gamma and interleukin (IL)-10 mRNA. There were significantly decreased IFN-gamma and tumour necrosis factor (TNF)-alpha mRNA levels, and a tendency toward higher levels of transforming growth factor-beta1 mRNA in HLA-DRB1*0301 positive compared with HLA-DRB1*0301 negative patients. Protein levels of IL-1beta, IL-2, IL-6, IL-12p70 and TNF-alpha in BALF were significantly higher in patients. HLA-DRB1*0301 positive patients exhibited tendencies to lower levels of most cytokines in BALF. In conclusion, the present data show a reduced expression of T-helper cell type-1 cytokines in human leukocyte-associated antigen-DRB1*0301 positive patients, which may relate to their good prognosis.

The human antimicrobial and chemotactic peptides LL-37 and alpha-defensins are expressed by specific lymphocyte and monocyte populations.

We identified antibacterial components in human T and natural killer (NK) cells by using freshly isolated lymphocytes enriched for T and NK cells as starting material. After growing these lymphocytes for 5 days in the presence of interleukin (IL)-2, we isolated and characterized several antibacterial peptides/proteins from the supernatant-alpha-defensins (HNP 1-3), LL-37, lysozyme, and a fragment of histone H2B-although other active components were also present. We then used reverse transcriptase-polymerase chain reaction to search for expression of the gene coding for LL-37 in several B-cell lines, gammadelta T-cell lines, NK clones, and one monocytic cell line, with positive results, but found no expression in several alphabeta T-cell lines. The alpha-defensins (HNP 1-3) were also found to be expressed in several of these cell lines. To confirm the presence of these antibacterial peptides in lymphocytes, we localized them to NK, gammadelta T cells, B cells, and monocytes/macrophages by using double-staining immunohistochemical analysis of freshly isolated lymphocytes. We also found that primary cultures of lymphocytes transcribe and secrete LL-37 and that these processes are affected by IL-6 and interferon-gamma. In addition, we demonstrated that LL-37 has chemotactic activity for polymorphonuclear leukocytes and CD4 T lymphocytes, whereas others have shown chemotactic activity for human alpha-defensins (HNP 1-2). These findings suggest that microbicidal peptides are effector molecules of lymphocytes and that antibacterial activity previously shown to be derived from T and NK cells may be partly mediated by the antibacterial peptides LL-37 and HNP 1-3.