IL-10 is an immunomodulatory cytokine with a critical role in limiting inflammation in immune-mediated pathologies. The mechanisms leading to IL-10 expression by CD4+ T cells are being elucidated, with several cytokines implicated. We explored the effect of IL-4 on the natural phenomenon of IL-10 production by a chronically stimulated antigen-specific population of differentiated Th1 cells. In vitro, IL-4 blockade inhibited while addition of exogenous IL-4 to Th1 cultures enhanced IL-10 production. In the in vivo setting of peptide immunotherapy leading to a chronically stimulated Th1 phenotype, lack of IL-4Rα inhibited the induction of IL-10. Exploring the interplay of Th1 and Th2 cells through co-culture, Th2-derived IL-4 promoted IL-10 expression by Th1 cultures, reducing their pathogenicity in vivo. Co-culture led to upregulated c-Maf expression with no decrease in the proportion of T-bet+ cells in these cultures. Addition of IL-4 also reduced the encephalitogenic capacity of Th1 cultures. These data demonstrate that IL-4 contributes to IL-10 production and that Th2 cells modulate Th1 cultures towards a self-regulatory phenotype, contributing to the cross-regulation of Th1 and Th2 cells. These findings are important in the context of Th1 driven diseases since they reveal how the Th1 phenotype and function can be modulated by IL-4.

Memory CD4+ T cells play a pivotal role in mediating long-term protective immunity, positioning them as an important target in vaccine development. However, multiple functionally distinct helper CD4+ T-cell subsets can arise in response to a single invading pathogen, complicating the identification of rare populations of memory precursor cells during the effector phase of infection and memory CD4+ T cells following pathogen clearance and the contraction phase of infection. Furthermore, current literature remains unclear regarding whether a single CD4+ memory T-cell lineage gives rise to secondary CD4+ T helper subsets or if there are unique memory precursor cells within each helper lineage. A majority of T follicular helper (Tfh) cells, which have established memory potential, express Id3, an inhibitor of E protein transcription factors, following acute viral infection. We show that expression of Id3 definitively identified a subset of cells within both the CD4+ Tfh and T helper 1 (Th1) lineages at memory time points that exhibited memory potential, with the capacity for significant re-expansion in response to secondary infection. Notably, we demonstrate that a subset of Th1 cells that survive into the memory phase were marked by Id3 expression and possessed the potential for enhanced expansion and generation of both Th1 and Tfh secondary effector cell populations in a secondary response to pathogen. Additionally, these cells exhibited enrichment of key molecules associated with memory potential when compared with Id3lo Th1 cells. Therefore, we propose that Id3 expression serves as an important marker to indicate multipotent potential in memory CD4+ T cells.

In addition to their use as colorants, anthraquinone derivatives have numerous medical applications, for example, as antibacterial and antiinflammatory agents. We confirmed that physcion (an anthraquinone derivative) induces TNF-alpha production by macrophages and increased the expressions of surface molecules (CD40, CD80, and CD86) and major histocompatibility complex (MHC) II. Based on these results, we hypothesized that physcion might induce the maturation of dendritic cells (DCs) to antigen-presenting cells (APCs), and decided to conduct in vitro experiments using bone-marrow-derived DCs (BMDCs). Physcion was not toxic to DCs and increased the expression of surface molecules (e.g., CD40, CD80, CD86, and MHC II) and the production of cytokines (e.g., IL-12p70, IL-1beta, IL-6, and TNF-alpha), but not of IL-10. To confirm that DCs matured by physcion induce T-cell-immune responses, naive CD4+ T cells were treated with physcion-treated DCs or their supernatants. Physcion induced the maturation of DCs, which promoted the polarization of Th1 cells. Our results show physcion-induced DC maturation via TLR4, and that mature DCs promote the differentiation of Th1 cells without affecting the differentiation of Th2 cells. These findings show that physcion has potential use as a treatment for inflammatory diseases associated with Th1/Th2 cell imbalance.

GLP-1 (glucagon-like peptide-1) has been reported to play a vital role in neuroprotection. Experimental autoimmune encephalomyelitis (EAE) is a well-established animal model widely used to study human multiple sclerosis, a chronic demyelination disease in the central nervous system (CNS). Recently, important studies have designated that the signaling axis of GLP-1 and its receptor controls the clinical manifestations and pathogenesis of EAE. However, it is elusive whether GLP-1 receptor signaling regulates the phenotype of autoreactive T cells in the CNS. We administered dulaglutide, a well-established GLP-1 receptor agonist (GLP-1 RA), to treat EAE mice prophylactically or semi-therapeutically and subsequently analyzed the mononuclear cells of the CNS. In this study, dulaglutide treatment significantly alleviates the clinical manifestations and histopathological outcomes of EAE. Dulaglutide decreases incidences of encephalitogenic Th1/Th17 cells and Th1 granulocyte-macrophage-colony-stimulating factor (GM-CSF) expression in the CNS. Administration of dulaglutide failed to control the chemotactic abilities of encephalitogenic Th1 and Th17 cells; however, prophylactic treatment considerably decreased the populations of dendritic cells and macrophages in the CNS parenchyma. These results obtained indicate that dulaglutide modulates the differentiation of encephalitogenic Th1/Th17 and the pathogenicity of Th1 cells by influencing antigen presenting cells quantities, providing mechanism insight on T cells regulation in ameliorating EAE by GLP-1.

T-cell characteristics are dynamic and influenced by multiple factors. To test whether cells and the environment in the central nervous system (CNS) can influence T-cells, we tested if culturing mouse CD4(+) T-cells on mouse primary astrocytes, compared with standard feeder cells, modified T-cell polarization to Th1 and Treg subtypes. Astrocytes supported the production of Th1 cells and Tregs, which was diminished by inflammatory activation of astrocytes, and glutamate accumulation that may result from impaired glutamate uptake by astrocytes strongly promoted Th1 production. These results demonstrate that astrocytes and the environment in the CNS have the capacity to regulate T-cell characteristics.

Precursor CD4+ T cells develop into effector Th1 and Th2 cells that play a central role in the immune response. We show that the JNK MAP kinase pathway is induced in Th1 but not in Th2 effector cells upon antigen stimulation. Further, the differentiation of precursor CD4+ T cells into effector Th1 but not Th2 cells is impaired in JNK2-deficient mice. The inability of IL-12 to differentiate JNK2-deficient CD4+ T cells fully into effector Th1 cells is caused by a defect in IFNgamma production during the early stages of differentiation. The addition of exogenous IFNgamma during differentiation restores IL-12-mediated Th1 polarization in the JNK2-deficient mice. The JNK MAP kinase signaling pathway, therefore, plays an important role in the balance of Th1 and Th2 immune responses.

Selective differentiation of CD4+ T helper (Th) cells into specialized subsets such as Th1 and Th2 cells is a key element of the adaptive immune system driving appropriate immune responses. Besides those canonical Th-cell lineages, hybrid phenotypes such as Th1/2 cells arise in vivo, and their generation could be reproduced in vitro. While master-regulator transcription factors like T-bet for Th1 and GATA-3 for Th2 cells drive and maintain differentiation into the canonical lineages, the transcriptional architecture of hybrid phenotypes is less well understood. In particular, it has remained unclear whether a hybrid phenotype implies a mixture of the effects of several canonical lineages for each gene, or rather a bimodal behavior across genes. Th-cell differentiation is a dynamic process in which the regulatory factors are modulated over time, but longitudinal studies of Th-cell differentiation are sparse. Here, we present a dynamic transcriptome analysis following Th-cell differentiation into Th1, Th2, and Th1/2 hybrid cells at 3-h time intervals in the first hours after stimulation. We identified an early bifurcation point in gene expression programs, and we found that only a minority of ~20% of Th cell-specific genes showed mixed effects from both Th1 and Th2 cells on Th1/2 hybrid cells. While most genes followed either Th1- or Th2-cell gene expression, another fraction of ~20% of genes followed a Th1 and Th2 cell-independent transcriptional program associated with the transcription factors STAT1 and STAT4. Overall, our results emphasize the key role of high-resolution longitudinal data for the characterization of cellular phenotypes.

Mechanisms of protective immunity to Staphylococcus aureus infection in humans remain elusive. While the importance of cellular immunity has been shown in mice, T cell responses in humans have not been characterised. Using a murine model of recurrent S. aureus peritonitis, we demonstrated that prior exposure to S. aureus enhanced IFNγ responses upon subsequent infection, while adoptive transfer of S. aureus antigen-specific Th1 cells was protective in naïve mice. Translating these findings, we found that S. aureus antigen-specific Th1 cells were also significantly expanded during human S. aureus bloodstream infection (BSI). These Th1 cells were CD45RO+, indicative of a memory phenotype. Thus, exposure to S. aureus induces memory Th1 cells in mice and humans, identifying Th1 cells as potential S. aureus vaccine targets. Consequently, we developed a model vaccine comprising staphylococcal clumping factor A, which we demonstrate to be an effective human T cell antigen, combined with the Th1-driving adjuvant CpG. This novel Th1-inducing vaccine conferred significant protection during S. aureus infection in mice. This study notably advances our understanding of S. aureus cellular immunity, and demonstrates for the first time that a correlate of S. aureus protective immunity identified in mice may be relevant in humans.

The chemokine receptor CXCR3, which was shown to take part in many inflammatory processes, is considered as a Th1 specific marker. Here, we show in a mouse model that CXCR3 expressing CD4(+) cells preferentially migrate to the peritoneal cavity under steady-state conditions. The peritoneal cavity milieu leads to an up-regulated expression of CXCR3. However, blocking of known ligands of this chemokine receptor did not alter the preferential migration. The peritoneal cavity environment also results in an increased percentage of memory cells producing cytokines. Up-regulation of IFNγ production occurs mostly in CXCR3(+) cells considered as Th1, whereas the up-regulation of IL-4 affects mostly in CXCR3(-) cells which are considered as Th2. We conclude that the peritoneal cavity does not change the Th-lineage of the cells, but that domination of this anatomic niche by Th1 cells rather results from preferential migration to this compartment.

Differentiated CD4(+) T cells preserve plasticity under various conditions. However, the stability of Th1 cells is unclear, as is whether Th1 cells can convert into Th17 cells and thereby contribute to the generation of IFN-γ(+) IL-17(+) CD4(+) T cells, the number of which correlates with severity of colitis. We investigated whether IFN-γ(+) Th1 cells can convert into Th17 cells under intestinal inflammation and the mechanisms involved. IFN-γ(Thy1.1+) Th1 cells were generated by culturing naïve CD4(+) T cells from IFN-γ(Thy1.1) CBir1 TCR-Tg reporter mice, whose TCR is specific for an immunodominant microbiota antigen, CBir1 flagellin, under Th1 polarizing conditions. IFN-γ(Thy1.1+) Th1 cells induced colitis in Rag(-/-) mice after adoptive transfer and converted into IL-17(+) Th17, but not Foxp3(+) Treg cells in the inflamed intestines. TGF-β and IL-6, but not IL-1β and IL-23, regulated Th1 conversion into Th17 cells. TGF-β induction of transcriptional factor Runx1 is crucial for the conversion, since silencing Runx1 by siRNA inhibited Th1 conversion into Th17 cells. Furthermore, TGF-β enhanced histone H3K9 acetylation but inhibited H3K9 trimethylation of Runx1- and ROR-γt-binding sites on il-17 or rorc gene in Th1 cells. We conclude that Th1 cells convert into Th17 cells under inflammatory conditions in intestines, which is possibly mediated by TGF-β induction of Runx1.

Exposure to Staphylococcus aureus enterotoxin B (SEB) is one of the causes of food poisoning and is associated with several immune diseases due to its superantigen capability. This study aimed to characterize the differentiations of naïve Th cells stimulated with different doses of SEB. The expression of T-bet, GATA-3, and Foxp3 or secretion of IFN-γ, IL-4, IL-5, IL-13, and IL-10 were evaluated in wild-type (WT) or DO11.10 CD4 T cells co-cultured with bone marrow dendritic cells (BMDCs). We found that the balance of Th1/Th2 could be dominated by the doses of SEB stimulation. A higher SEB dose could induce more Th1 and a lower Th2/Th1 ratio in Th cells co-cultured with BMDCs. This different tendency of Th cell differentiation induced by the SEB complements the existing knowledge about SEB acting as a superantigen to activate Th cells. Additionally, it is also helpful in managing the colonization of S. aureus and food contamination of SEB.

The central role of tumor-specific TH1 cells in anticancer immune responses is becoming increasingly appreciated. However, little is known about how these cells are generated in vivo. Here, we used flow cytometry and gene expression microarrays to characterize the primary activation and TH1 differentiation of naïve tumor-specific CD4+ T cells in a mouse model of cancer immunosurveillance. We took advantage of T-cell receptor-transgenic mice in which CD4+ T cells recognize a tumor-specific antigen secreted by MHC class II-negative MOPC315 myeloma cells. Cancer cells were injected subcutaneously and T-cell activation was analyzed in draining lymph nodes and at the incipient tumor site 8 d later. Upon activation and migration to incipient tumor sites, tumor-specific CD4+ T cells exhibited the upregulation of 29 cell-surface molecules (CD2, CD5, CD11a, CD18, CD25, CD28, CD44, CD45, CD49d, CD51, CD54, CD69, CD71, CD83, CD86, CD90, CD95, CD102, CD122, CD153, CD166, CD200, CD249, CD254, CD274, CD279, Ly6C, MHC class I and CCR7) and the downregulation of five (CD27, CD31, CD45RB, CD62L and CD126). Activated CD4+ T cells produced interferon γ, a cytokine consistent with a TH1-polarized response, tumor necrosis factor α as well as interleukin (IL)-2, IL-3 and IL-10. The activation of naïve tumor-specific CD4+ T cells in draining lymph nodes resulted in the upregulation of 609 genes and the downregulation of 284 genes. The bioinformatic analysis of differentially expressed genes identified functional pathways related to tumor-specific TH1 cell activation. This study may represent a useful resource to guide the development of TH1-based immunotherapies against cancer.

Primary and secondary (boosted) memory CD8 T cells exhibit differences in gene expression, phenotype and function. The impact of repeated antigen stimulations on memory CD4 T cells is largely unknown. To address this issue, we utilized LCMV and Listeria monocytogenes infection of mice to characterize primary and secondary antigen (Ag)-specific Th1 CD4 T cell responses. Ag-specific primary memory CD4 T cells display a CD62L(lo)CCR7(hi) CD27(hi) CD127(hi) phenotype and are polyfunctional (most produce IFNγ, TNFα and IL-2). Following homologous prime-boost immunization we observed pathogen-specific differences in the rate of CD62L and CCR7 upregulation on memory CD4 T cells as well as in IL-2+IFNγco-production by secondary effectors. Phenotypic and functional plasticity of memory Th1 cells was observed following heterologous prime-boost immunization, wherein secondary memory CD4 T cells acquired phenotypic and functional characteristics dictated by the boosting agent rather than the primary immunizing agent. Our data also demonstrate that secondary memory Th1 cells accelerated neutralizing Ab formation in response to LCMV infection, suggesting enhanced capacity of this population to provide quality help for antibody production. Collectively these data have important implications for prime-boost vaccination strategies that seek to enhance protective immune responses mediated by Th1 CD4 T cell responses.

Sulfatides have immunomodulatory functions, and play protective roles in multiple autoimmune diseases. In the present study, we showed that sulfatides ameliorated experimental autoimmune neuritis in Lewis rats induced with bovine peripheral myelin, which was associated with decreased proportions of Th1 and Th17 cells. Furthermore, compared control group, cells from sulfatide-treated rats exhibited lower potential in proliferation and IL-17 secretion in the presence of BPM or ConA in vitro. Moreover, sulfatides also reduced the proportions of NK and NKT cells. In summary, our study indicated that sulfatides might become a new therapeutic agent in Guillain-Barré syndrome in the future.

The junctional adhesion molecule-A (JAM-A) is an adhesion molecule present in the surface of several cell types, such as endothelial cells and leukocytes as well as Dendritic Cells (DC). Given the potential relevance of JAM-A in diverse pathological conditions such as inflammatory diseases and cancer, we investigated the role of JAM-A in CD4+ T cell priming. We demonstrate that JAM-A is present in the immunological synapse formed between T cells and DC during priming. Furthermore, an antagonistic anti-JAM-A mAb could disrupt the interaction between CD4+ T cell and DC. Antagonism of JAM-A also attenuated T cell activation and proliferation with a decrease in T-bet expression and increased IL-6 and IL-17 secretion. These findings demonstrate a functional role for JAM-A in interactions between CD4+ T cells and DCs during T cell priming as a positive regulator of Th1 differentiation.

Adipose tissue-derived stem cells (ADSC) have been shown to possess stem cell properties such as transdifferentiation, self-renewal and therapeutic potential. However, the property of ADSC to accommodate immune system is still unknown. In this study, ADSC were cocultured with allogenetic dendritic cells (DC), and then treated DC were mixed with allogenetic CD4+ T cells. The results demonstrated that ADSC could downregulate costimulatory molecules, including CD80, CD83, CD86, and cytokine secretion such as interleukin (IL)-12 and tumor necrosis factor (TNF)-α, while upregulate indoleamine-2,3-dioxygenase (IDO) of allogenetic DC. In addition, treated DC could inhibit CD4+ T cell activation and naïve T cells toward Th1 polarization. The results suggest that ADSC could negatively modulate immunity and induce immune tolerance, which provide a promising strategy in transplantation or autoimmune disease.

The HER family of receptor tyrosine kinases has been linked to deregulation of growth and proliferation for multiple types of cancer. Members have therefore become thefocus of many drug and immune-based therapy innovations. The targeted anti-cancer agent, lapatinib, is a small molecule inhibitor that directly interferes with EGFR (HER-1)and HER-2 signaling, and indirectly reduces HER-3 signaling, thus suppressing important downstream events. A recently-developed dendritic cell-based vaccine against early breast cancer (ductal carcinoma in situ; DCIS) that generates strong Th1-dominated immunity against HER-2 has induced pathologic complete response in about one-third of immunized individuals. In vitro studies suggested cytokines secreted by Th1 cells could be major contributors to the vaccine effects including induction of apoptosis and suppression of HER expression. With a view toward improving complete response rates, we investigated whether the principle Th1 cytokines (IFN-γ and TNF-α) could act in concert with lapatinib to suppress activity of breast cancer lines in vitro. Lapatinib-sensitive SKBR3, MDA-MB-468 and BT474 cells were incubated with Th1 cytokines, lapatinib, or both. It was found that combined treatment maximized metabolic suppression(Alamar Blue assay), as well as cell death (Trypan Blue) and apoptosis(Annexin V/Propidium Iodide and TMRE staining). Combined drug plus cytokine treatment also maximized suppression of both total and phosphorylated forms of HER-2 and HER-3. Interestingly, when lapatinib resistant lines MDA-MB-453 and JIMT-1 were tested, it was found that the presence of Th1 cytokines appeared to enhance sensitivity for lapatinib-induced metabolic suppression and induction of apoptotic cell death, nearly abrogating drug resistance. These studies provide pre-clinical data suggesting the possibility that targeted drug therapy may be combined with vaccination to enhance anti-cancer effects, and furthermore that robust immunity in the form of secreted Th1 cytokines may have the capacity to mitigate resistance to targeted drugs.

CD4+ T cells that co-express CD25 and CD127 (CD25+CD127+) make up around 20% of all circulating CD4+ memory T cells in healthy people. The clinical significance of these cells is that in children with type 1 diabetes their relative frequency at diagnosis is significantly and directly correlated with rate of disease progression. The purpose of this study was to further characterize the CD25+CD127hi cells. We show that they are a mix of Th1 and Th2 cells however, they have a significantly higher relative frequency of pre-committed and committed Th2 cells, and secrete significantly higher levels of Th2-type cytokines than CD25- memory T cells. Further, these cells are neither exhausted nor senescent and proliferate to the same extent as CD25- memory cells. Thus, CD25+CD127hi cells are a highly active subset of memory T cells that might play a role in controlling inflammation via anti-inflammatory Th2-type deviation.

Although the importance of cytotoxic T lymphocytes and neutralizing antibodies for antiviral defense is well known, the antiviral mechanism of Th1 remains unclear. We show that Th1 cells mediate noncytolytic antiviral protection independent of direct lysis through local secretion of IFN-gamma after herpes simplex virus (HSV) 2 infection. IFN-gamma acted on stromal cells, but not on hematopoietic cells, to prevent further viral replication and spread throughout the vaginal mucosa. Importantly, unlike other known Th1 defense mechanisms, this effector function did not require recognition of virally infected cells via MHC class II. Instead, recall Th1 response was elicited by MHC class II(+) antigen-presenting cells at the site of infection. Dendritic cells (DCs) were not required and only partially sufficient to induce a recall response from memory Th1 cells. Importantly, DCs and B cells together contributed to restimulating memory CD4 T cells to secrete IFN-gamma. In the absence of both DCs and B cells, immunized mice rapidly succumbed to HSV-2 infection and death. Thus, these results revealed a distinct mechanism by which memory Th1 cells mediate noncytolytic IFN-gamma-dependent antiviral protection after recognition of processed viral antigens by local DCs and B cells.

To evaluate the Th1/Th2 cell profile in spleens of cirrhotic and hypersplenic rats by investigating the expression of Th1-associated chemokine receptors CXCR3, CCR5 and Th2-associated chemokine receptor CCR3.