A multivariate Th17 metagene for prognostic stratification in T cell non-inflamed triple negative breast cancer.
ABSTRACT: A diversity of T helper (Th) subsets (Th1, Th2, Th17) has been identified in the human tumor microenvironment. In breast cancer, the role of Th subsets remains controversial, and a systematic study integrating Th subset diversity, T cell inflammation, breast cancer molecular subtypes, and patient prognosis, is lacking. In primary untreated breast cancer samples, we analyzed 19 Th cytokines at the protein level. Eight were T cell-specific, and subsequently measured in 106 prospectively-collected untreated samples. The dominant Th cytokines across all breast cancer samples were IFN-? and IL-2. Th2 cytokines (IL-4, IL-5, IL-13) were expressed at low levels and not associated with any breast cancer subtype. Th17 cytokines (IL-17A and IL-17F) were up-regulated in triple negative breast cancer (TNBC), specifically in T cell non-inflamed tumors. In order to get insight into prognosis, we exploited the METABRIC transcriptomic dataset. We derived Th1, Th2, and Th17 metagenes based on manually curated Th signatures, and found that a high Th17 metagene was of good prognosis in T cell non-inflamed TNBC. Multivariate Cox modeling selected the Nottingham Prognostic Index (NPI), Th2 and Th17 metagenes as additive predictors of breast cancer-specific survival, which defined novel and highly distinct prognostic groups within TNBC. Our results reveal that Th17 is a novel prognostic composite biomarker in T cell non-inflamed TNBC. Integrating immune cell and tumor molecular diversity is an efficient strategy for prognostic stratification of cancer patients.
Project description:Immune signatures in breast tumors differ by estrogen receptor (ER) status. The purpose of this study was to assess associations between ER phenotypes and circulating levels of cytokines that co-ordinate cell-mediated [T-helper type 1 (Th1)] and humoral [T-helper type 2 (Th2)] immunity. We conducted a case-case comparison of 523 women with newly diagnosed breast cancer to evaluate associations between 27 circulating cytokines, measured using Luminex XMap technology, and breast cancer phenotypes [ER(-) vs. ER(+); triple negative breast cancer (TNBC) vs. luminal A (LumA)]. Ratios of Th1 to Th2 cytokines were also evaluated. Levels of interleukin (IL)-5, a Th-2 cytokine, were higher in ER(-) than in ER(+) tumors. The highest tertile of IL-5 was more strongly associated with ER(-) (OR = 2.33, 95 % CI 1.40-3.90) and TNBCs (OR = 2.78, 95 % CI 1.53-5.06) compared to ER(+) and LumA cancers, respectively, particularly among premenopausal women (OR = 4.17, 95 % CI 1.86-9.34, ER(-) vs. ER(+); OR = 5.60, 95 % CI 2.09-15.01, TNBC vs. LumA). Elevated Th1 cytokines were also detected in women with ER(-) and TNBCs, with women in the highest tertile of interferon ?2 (OR = 2.39, 95 % CI 1.31-4.35) or tumor necrosis factor-? (OR = 2.27, 95 % CI 1.21-4.26) being twice as likely to have TNBC versus LumA cancer. When cytokine ratios were examined, women with the highest ratios of Th1 cytokines to IL-5 levels were least likely to have ER(-) or TNBCs compared to ER(+) or LumA cancers, respectively. The strongest associations were in premenopausal women, who were up to 80 % less likely to have TNBC than LumA cancers (IL-12p40/IL-5, OR = 0.19, 95 % CI 0.07-0.56). These findings indicate that immune function is associated with ER(-) and TNBC and may be most relevant among younger women, who are likely to be diagnosed with these aggressive phenotypes.
Project description:In chronically inflamed tissues, such as those affected by autoimmune disease, activated Th cells often colocalize with monocytes. We investigate in this study how murine Th cells influence the phenotype and function of monocytes. The data demonstrate that Th1, Th2, and Th17 subsets promote the differentiation of autologous monocytes into MHC class II(+), CD11b(+), CD11c(+) DC that we call DCTh. Although all Th subsets induce the formation of DCTh, activated Th17 cells uniquely promote the formation of IL-12/IL-23-producing DCTh (DCTh17) that can polarize both naive and Th17 cells to a Th1 phenotype. In the inflamed CNS of mice with Th17-mediated experimental autoimmune encephalomyelitis, Th cells colocalize with DC, as well as monocytes, and the Th cells obtained from these lesions drive the formation of DCTh that are phenotypically indistinguishable from DCTh17 and polarize naive T cells toward a Th1 phenotype. These results suggest that DCTh17 are critical in the interplay of Th17- and Th1-mediated responses and may explain the previous finding that IL-17-secreting Th cells become IFN-?-secreting Th1 cells in experimental autoimmune encephalomyelitis and other autoimmune disorders.
Project description:INTRODUCTION: Current prognostic gene expression profiles for breast cancer mainly reflect proliferation status and are most useful in ER-positive cancers. Triple negative breast cancers (TNBC) are clinically heterogeneous and prognostic markers and biology-based therapies are needed to better treat this disease. METHODS: We assembled Affymetrix gene expression data for 579 TNBC and performed unsupervised analysis to define metagenes that distinguish molecular subsets within TNBC. We used n = 394 cases for discovery and n = 185 cases for validation. Sixteen metagenes emerged that identified basal-like, apocrine and claudin-low molecular subtypes, or reflected various non-neoplastic cell populations, including immune cells, blood, adipocytes, stroma, angiogenesis and inflammation within the cancer. The expressions of these metagenes were correlated with survival and multivariate analysis was performed, including routine clinical and pathological variables. RESULTS: Seventy-three percent of TNBC displayed basal-like molecular subtype that correlated with high histological grade and younger age. Survival of basal-like TNBC was not different from non basal-like TNBC. High expression of immune cell metagenes was associated with good and high expression of inflammation and angiogenesis-related metagenes were associated with poor prognosis. A ratio of high B-cell and low IL-8 metagenes identified 32% of TNBC with good prognosis (hazard ratio (HR) 0.37, 95% CI 0.22 to 0.61; P < 0.001) and was the only significant predictor in multivariate analysis including routine clinicopathological variables. CONCLUSIONS: We describe a ratio of high B-cell presence and low IL-8 activity as a powerful new prognostic marker for TNBC. Inhibition of the IL-8 pathway also represents an attractive novel therapeutic target for this disease.
Project description:Tuberculosis (TB) is a major health problem of global concern. The control of this disease requires appropriate preventive measures, including vaccines. In TB, T helper (Th)1 cytokines provide protection whereas Th2 and T regulatory (Treg) cytokines contribute to the pathogenesis and Th17 cytokines play a role in both protection and pathogenesis. Previous studies with Mycobacterium tuberculosis-specific proteins have identified seven low molecular weight proteins, PE35, ESXA, ESXB, Rv2346c, Rv2347c, Rv3619c, and Rv3620c, as immunodominant antigens inducing Th1-cell responses in humans following natural infection with M. tuberculosis. The aim of this study was to characterize the cytokine responses induced in mice immunized with these proteins, using various adjuvants and delivery systems, i.e. chemical adjuvants (Alum and IFA), non-pathogenic mycobacteria (M. smegmatis and M. vaccae) and a DNA vaccine plasmid (pUMVC6). The immune responses were monitored by quantifying the marker cytokines secreted by Th1 (IFN-?), Th2 (IL-5), Treg (IL-10), and Th17 (IL-17A) cells. DNA corresponding to pe35, esxa, esxb, rv2346c, rv2347c, rv3619c, and rv3620c genes were cloned into the expression vectors pGES-TH-1, pDE22 and pUMVC6 for expression in Escherichia coli, mycobacteria and eukaryotic cells, respectively. Mice were immunized with the recombinants using different adjuvants and delivery systems, and spleen cells were stimulated in vitro with peptides of immunizing proteins to investigate antigen-specific secretion of Th1 (IFN-?), Th2 (IL-5), Treg (IL-10), and Th17 (IL-17A) cytokines. The results showed that spleen cells, from mice immunized with all antigens, secreted the protective Th1 cytokine IFN-?, except ESXB, with one or more adjuvants and delivery systems. However, only Rv3619c consistently induced Th1-biased responses, without the secretion of significant concentrations of Th2, Th17 and Treg cytokines, with all adjuvants and delivery systems. Rv3619c also induced antigen-specific IgG antibodies in immunized mice.
Project description:Atopic dermatitis (AD) is the most common chronic and relapsing inflammatory skin disease. AD is typically characterized by skewed T helper (Th) 2 inflammation, yet other inflammatory profiles (Th1, Th17, Th22) have been observed in human patients. How cytokines from these different Th subsets impact barrier function in this disease is not well understood. As such, we investigated the impact of the canonical Th17 cytokine, IL-17A, on barrier function and protein composition in primary human keratinocytes and human skin explants. These studies demonstrated that IL-17A enhanced tight junction formation and function in both systems, with a dependence on STAT3 signaling. Importantly, the Th2 cytokine, IL-4 inhibited the barrier-enhancing effect of IL-17A treatment. These observations propose that IL-17A helps to restore skin barrier function, but this action is antagonized by Th2 cytokines. This suggests that restoration of IL-17/IL-4 ratio in the skin of AD patients may improve barrier function and in so doing improve disease severity.
Project description:Neuromuscular transmission failure in myasthenia gravis (MG) is most commonly elicited by autoantibodies (ab) to the acetylcholine receptor or the muscle-specific kinase, constituting AChR-MG and MuSK-MG. It is controversial whether these MG subtypes arise through different T helper (Th) 1, Th2 or Th17 polarized immune reactions and how these reactions are blunted by immunosuppression. To address these questions, plasma levels of cytokines related to various Th subtypes were determined in patients with AChR-MG, MuSK-MG and healthy controls (CON). Peripheral blood mononuclear cells (PBMC) were activated in vitro by anti-CD3, and cytokines were quantified in supernatants. In purified blood CD4+ T cells, RNA of various cytokines, Th subtype specific transcription factors and the co-stimulatory molecule, CD40L, were quantified by qRT-PCR. Plasma levels of Th1, Th2 and Th17 related cytokines were overall not significantly different between MG subtypes and CON. By contrast, in vitro stimulated PBMC from MuSK-MG but not AChR-MG patients showed significantly increased secretion of the Th1, Th17 and T follicular helper cell related cytokines, IFN-?, IL-17A and IL-21. Stimulated expression of IL-4, IL-6, IL-10 and IL-13 was not significantly different. At the RNA level, expression of CD40L by CD4+ T cells was reduced in both AChR-MG and MuSK-MG patients while expression of Th subset related cytokines and transcription factors were normal. Immunosuppression treatment had two effects: First, it reduced levels of IL12p40 in the plasma of AChR-MG and MuSK-MG patients, leaving other cytokine levels unchanged; second, it reduced spontaneous secretion of IFN-? and increased secretion of IL-6 and IL-10 by cultured PBMC from AChR-MG, but not MuSK-MG patients. We conclude that Th1 and Th17 immune reactions play a role in MuSK-MG. Immunosuppression attenuates the Th1 response in AChR-MG and MuSK-MG, but otherwise modulates immune responses in AChR-MG and MuSK-MG patients differentially.
Project description:T helper (Th) 17 cells produce the effector cytokine interleukin (IL)-17, along with IL-22, which stimulates colonic epithelial cells to produce a membrane-bound mucin, Muc1. Muc1 is a component of the colonic mucus, which functions as a lubricant and a physiologic barrier between luminal contents and mucosal surface. The gene MUC1 has been associated with susceptibility to inflammatory bowel disease; we investigated the role of Muc1 in development of colitis in mice.Muc1 and RAG1 were disrupted in mice (Muc/RAG double knockout mice); Th1-mediated colitis was induced by intravenous injection of CD4(+)CD45RB(high) T cells. We also studied Th2-mediated colitis using mice with disruptions in Muc1 and T-cell receptor ? chain (Muc/TCR double knockout mice).Muc1 deficiency led to the development of more severe forms of Th1- and Th2-induced colitis than controls. Loss of Muc1 increased colonic permeability and the Th17-cell, but not Th2 or Th1 cell, response in the inflamed colon. Loss of Muc1 also promoted expansion of an innate lymphoid cell population (Lin(-) ckit(-) Thy1(+) Sca1(+)) that produces IL-17. The expansion of Th17 adaptive immune cells and innate lymphoid cells required the commensal microbiota.Muc1, which is up-regulated by Th17 signaling, functions in a negative feedback pathway that prevents an excessive Th17 cell response in inflamed colons of mice. Disruption of this negative feedback pathway, perhaps by variants in Muc1, might contribute to inflammatory bowel disease in patients.
Project description:BACKGROUND:The majority of CD4+ T helper (Th) cells found in the synovial fluid (SF) of patients with rheumatoid arthritis (RA) express CXCR3, a receptor associated with Th1 cells. In blood, subsets of Th2 and Th17 cells also express CXCR3, but it is unknown if these cells are present in RA SF or how cytokines from these subsets affect cytokine/chemokine secretion by fibroblast-like synoviocytes (FLS) from patients with RA. METHODS:We examined the proportions of Th1, Th2, CXCR3+Th2, Th17, CXCR3+Th17, Th1Th17, peripheral T helper (TPh) and T follicular helper (TFh) cells in paired SF and blood, as well as the phenotype of TPh and TFh cells in RA SF (n?=?8), by the use of flow cytometry. We also examined the cytokine/chemokine profile in paired SF and plasma (n?=?8) and in culture supernatants of FLS from patients with chronic RA (n?=?7) stimulated with Th-associated cytokines, by the use of cytometric bead arrays and ELISA. Cytokine receptor expression in FLS (n?=?3) were assessed by the use of RNA sequencing and qPCR. RESULTS:The proportions of Th1 and CXCR3+Th2 cells were higher in SF than in blood (P?<?0.05). TPh and PD-1highTFh in RA SF were primarily of a Th1 and a CXCR3+Th2 phenotype. Moreover, the levels of CXCL9, CXCL10, CCL20, CCL2, CXCL8, IL-6 and IL-10 were higher in SF than in plasma (P?<?0.05). Lastly, IL-4, IL-13 and IL-17A induced RA FLS to secrete proinflammatory IL-6, CCL2, CXCL1 and CXCL8, while IFN? mainly induced CXCL10. CONCLUSION:These findings indicate that not only Th1 but also CXCR3+Th2 cells may have a pathogenic role in RA synovial inflammation.
Project description:Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype lacking expression of estrogen and progesterone receptors (ER/PR) and HER2, thus limiting therapy options. We hypothesized that meta-analysis of TNBC gene expression profiles would illuminate mechanisms underlying the aggressive nature of this disease and identify therapeutic targets. Meta-analysis in the Oncomine database identified 206 genes that were recurrently deregulated in TNBC compared with non-TNBC and in tumors that metastasized or led to death within 5 years. This 'aggressiveness gene list' was enriched for two core functions/metagenes: chromosomal instability (CIN) and ER signaling metagenes. We calculated an 'aggressiveness score' as the ratio of the CIN metagene to the ER metagene, which identified aggressive tumors in breast cancer data sets regardless of subtype or other clinico-pathological indicators. A score calculated from six genes from the CIN metagene and two genes from the ER metagene recapitulated the aggressiveness score. By multivariate survival analysis, we show that our aggressiveness scores (from 206 genes or the 8 representative genes) outperformed several published prognostic signatures. Small interfering RNA screen revealed that the CIN metagene holds therapeutic targets against TNBC. Particularly, the inhibition of TTK significantly reduced the survival of TNBC cells and synergized with docetaxel in vitro. Importantly, mitosis-independent expression of TTK protein was associated with aggressive subgroups, poor survival and further stratified outcome within grade 3, lymph node-positive, HER2-positive and TNBC patients. In conclusion, we identified the core components of CIN and ER metagenes that identify aggressive breast tumors and have therapeutic potential in TNBC and aggressive breast tumors. Prognostication from these metagenes at the mRNA level was limited to ER-positive tumors. However, we provide evidence that mitosis-independent expression of TTK protein was prognostic in TNBC and other aggressive breast cancer subgroups, suggesting that protection of CIN/aneuploidy drives aggressiveness and treatment resistance.
Project description:IL-17 is the signature cytokine of recently discovered Th type 17 (Th17) cells, which are prominent in defense against extracellular bacteria and fungi as well as in autoimmune diseases, such as rheumatoid arthritis and experimental autoimmune encephalomyelitis in animal models. IL-25 is a member of the IL-17 family of cytokines, but has been associated with Th2 responses instead and may negatively cross-regulate Th17/IL-17 responses. IL-25 can initiate an allergic asthma-like inflammation in the airways, which includes recruitment of eosinophils, mucus hypersecretion, Th2 cytokine production, and airways hyperreactivity. We demonstrate that these effects of IL-25 are entirely dependent on the adaptor protein CIKS (also known as Act1). Surprisingly, this adaptor is necessary to transmit IL-17 signals as well, despite the very distinct biologic responses that these two cytokines elicit. We identify CD11c(+) macrophage-like lung cells as physiologic relevant targets of IL-25 in vivo.