Search

The development of an immunogenic, effective, and safe vaccine is essential as an alternative for disease control. The present study aimed to evaluate the immunogenicity and efficacy potential of a polyepitope T-cell antigen candidate against visceral leishmaniasis in a murine model. BALB/c mice were immunized with three doses subcutaneously with Poly-T Leish alone or adjuvanted with Saponin plus Monophosphoryl lipid A, with 15-day intervals between doses, and challenged with 10

Tissue-resident memory T (Trm) cells can trigger a secondary immune response when they encounter the same antigen, playing an important role in antitumor immunity. However, whether Trm cells are protective against female genital tract tumors remainunknown. Here, we show that cervicovaginal vaccination with HPV16 E7aa

CD40-CD40L and inducible co-stimulatory molecule (ICOS)-ICOSL ligations are demonstrated to play critical roles in CD4+T-B interaction for B cell activation and differentiation in mouse models. Herein, by using a micropipette adhesion assay and an in vitro CD4+T-B cell coculture system simultaneously, we intended to dissect their roles in human CD4+T-B adhesion and IgG/IgM production. With the upregulation of CD40L and ICOS expressions on CD4+ T cells upon TCR/CD28 stimulation in vitro, activated CD4+ T cells exhibited enhanced adhesion with autologous B cells at a single cell level when compared to the resting counterparts. Blockade of ICOS dramatically damped the adhesion between CD4+ T and B cells whereas less effect of CD40L blockade was observed. On the contrary, blockade of CD40L led to the dramatic decrease in IgG/IgM production when B cells were cocultured with activated CD4+ T cells together with the decrease in the induction of CD19hi B cells. However, ICOS blockade displayed less attenuation on IgG/IgM production. Distinct roles of CD40-CD40L and ICOS-ICOSL in cell adhesion and IgG/IgM production were also observed in CD4+T-B cell interaction in system lupus erythematosus patients. The blockade of CD40L, rather than ICOS, led to the dramatic decrease in the phosphorylation of Pyk2 in CD19hi B cells and total B cells. Our study thus provides the evidence that CD40L and ICOS on activated CD4+ T cells either upon in vitro activation or at the pathogenic state function diversely during CD4+T-B cell interactions. While ICOS-ICOSL ligation is more likely to be engaged in cell adhesion, CD40-CD40L provides indispensable signal for B cell differentiation and IgG/IgM production. Our results are thus indicative for the segregating costimulation of CD40-CD40L and ICOS-ICOSL on CD4+ T cells for B cell activation and differentiation, which might be helpful for the dissection of SLE pathogenesis.

MAIT cells are unconventional innate-like T lymphocytes contributing to host immune protection against Mycobacteria tuberculosis (Mtb) infection. CD4

For over 70 years experimental autoimmune encephalomyelitis (EAE) has been induced with myelin autoantigens emulsified in complete Freund’s adjuvant (CFA) which has significant side effects such as pain, inflammation, and tissue necrosis at the injection site. β-1,3- d -glucan particles (GPs) are hollow microcapsules prepared from Saccharomyces cerevisiae cell walls that induce potent Th17 cell responses without causing strong injection site tissue reactions. We evaluated the potential of GPs complexed with neuroantigens to induce EAE while avoiding undesirable side effects. GPs loaded with myelin oligodendrocyte glycoprotein 35–55 (MOG35–55) or proteolipid protein 139–151 (PLP139–151) peptides effectively induced EAE in C57BL/6 mice and SJL mice. Disease severity, CNS pathology and immune responses were comparable between GP- and CFA-immunized mice. Importantly, injection with GPs resulted in significantly decreased inflammation compared with CFA. We posit that use of GPs provides an alternative means for inducing EAE that results in comparable disease, but less discomfort to animals.

Previously, we generated IL233 - a hybrid cytokine composed of interleukin (IL)-2 and IL-33, with better therapeutic potential than either cytokine in multiple inflammatory diseases, in part through promoting T-regulatory cells (Tregs). Here we test the potential of IL233 pretreatment in a murine model of excessive Th1 activation, the parent-into-F1 model of acute GVHD (aGVHD). Five days of IL233 pretreatment of the recipients blocked or delayed the aGVHD-linked loss of B-cells as seen in either the peripheral blood (day-11) or lymph nodes (day-14). IL233 pretreatment also prevented the expansion of donor CD8 T-cells in blood and LN at day-14 and significantly reduced day-14 serum IFNγ and TNFγ compared to saline treated GVHD mice although, the level of Tregs did not statistically differ between saline and IL233-treated mice. Overall, the current study provides support for the use of IL233 as a therapeutic option in excessive Th1/CD8-driven conditions.

The imbalance of helper T (Th) 17 and regulatory T (Treg) cells plays an important role in the pathogenesis of pneumonia. This study aims to investigate the role and mechanism of long non-coding RNA growth arrest-specific 5 (GAS5) in the differentiation of Th17 cells and Tregs in childhood pneumonia. Expression of GAS5, miR-217, signal transducer and activator of transcription-5 (STAT5), receptor-related orphan receptor γt (RORγt), and transcription factor Forkhead box P3 (Foxp3) were examined by qRT-PCR and western blot. The percentage of Th17 cells and Tregs in CD4+ T cells were measured by flow cytometry. The interaction between miR-217 and GAS5 or STAT5 was analyzed by luciferase reporter assay. Downregulated GAS5 expression and Treg cell percentage, and upregulated Th17 cell percentage were observed in pneumonia patients when compared with the healthy controls. Furthermore, GAS5 overexpression corrected the imbalanced Th17/Treg in peripheral blood CD4+ T cells derived from pneumonia patients, and this effect was reversed by miR-217 mimic and STAT5 silencing. Mechanistically, GAS5 acted as a sponge of miR-217 to reduce binding of miR-217 to its target STAT5, leading to upregulation of STAT5 expression. Taken together, GAS5 corrects the Treg/Th17 imbalance by targeting the miR-217/STAT5 axis in childhood pneumonia.

Although contributions of IL-33 to pulmonary diseases, including asthma, have been well documented, the complexity of such regulation warrants additional exploration. To better understand the involvement of IL-33, we used a murine asthma surrogate based on sensitisation and challenge with dust mite extract in the presence/absence of IL-33. Murine models were established with Dermatophagoides farinae (Der f) to establish (1) the effect of co-administered rmIL-33; (2) the effect of prior glucocorticoid intervention; (3) the effect of IL-33 on challenge with sub-threshold dosage Der f. The effects of rmIL-33 on bone marrow-derived dendritic cells were explored in vitro. Mice challenged with Der f combined with IL-33 compared with diluent control evinced significantly more airways inflammation and local cytokine production which was less sensitive to inhibition by dexamethasone. IL-33 also induced airways hyperresponsiveness, eosinophilic inflammation and cytokine production in lung tissues of animals exposed to sub-threshold dosage of Der f. In vitro, IL-33-stimulated DCs showed a significantly elevated capacity to stimulate CD4+ T cell proliferation and cytokine production and were also significantly more resistant to dexamethasone-induced apoptosis. Our data suggest that IL-33 reduces the threshold for allergen-induced inflammation of the airways in acorticosteroid-resistant fashion possibly in part through acting on DCs, a phenomenon which may be relevant to the development of severe, corticosteroid-resistant airways obstruction in human asthmatic patients.

The liver contains a rich mix of T cells, including activated T cells, tissue-resident memory T cells and cells undergoing apoptosis. When antigens are presented in this milieu the default result is functional tolerance. T cell tolerance in the liver could be constitutive, or it could be adaptive, in which case liver cells would become unresponsive after encountering antigen in the liver context. To test this model, we evaluated the potential of human liver T cells to respond to T cell receptor ligation in liver tissue slice cultures. These T cells contained an actively motile subset of CD4+ T cells marked by CCR7 and CD62L, and fully functional subsets of CD4+ and CD8+ T cells that synthesized effector cytokines but subsequently assumed an exhausted phenotype. These data favor the model that human liver T cells are not constitutively tolerant but undergo adaptive tolerance after activation.

B cells play a crucial role in immune responses. The main functions include B cell protective antibody production, inflammation reduction, activation and proliferation. Long non-coding RNAs (lncRNAs) have been reported to act as important regulators of many pathological processes. However, few lncRNAs have been reported to affect B cell function. In this study, we explored the expression and role of lncRNA 2900052N01Rik (lnc-290) in lipopolysaccharide (LPS)-induced B cells purified from mouse spleens in vitro. Here, we confirmed that lnc-290 was highly expressed in B cells stimulated by LPS. Knockdown of lnc-290 inhibited the expression of CD69/CD86 and the growth of B cells. Moreover, down-regulated lnc-290 reduced B cell differentiation and immunoglobulin production in vitro. In addition, we found that lnc-290 regulated LPS-induced B cell activation via the NF-κB/ERK pathways. Interestingly, abnormal lnc-290 expression did not alter the B cell activation or proliferation induced by IL-4 or CD40/CD40L. Accordingly, these results indicated, for the first time, that lnc-290 down-regulation inhibits LPS-induced B cell proliferation, activation and differentiation by blocking the LPS/TLR4 signaling pathway. Together, the in vitro data demonstrate that lnc-290 participated in the inflammation and tissue damage mediated by LPS-activated B cells.

Efficacy of B-cell depletion therapy highlights the antibody-independent effector functions of B cells in rheumatoid arthritis (RA). Given type 1 helper T (Th1) cells abundant in synovial fluid (SF) of RA, we have determined whether Th1 cells could generate novel effector B cells. Microarray and qPCR analysis identified CXCL9/10 transcripts as highly expressed genes upon BCR/CD40/IFN-γ stimulation. Activated Th1 cells promoted the generation of CXCL9/10-producing T-bet

Systemic lupus erythematosus (SLE) is an autoimmune disease leading to considerable morbidity worldwide, which can be developed from a breakdown in immunological tolerance, resulting in T cell hyperactivation. T cell hyperactivation has been implicated in the tissue damage associated with many diseases. Although many researchers have identified the involvement of T-cell receptor-associated signaling molecules in T-cell activation, the mechanisms underlying this process are yet to be elaborated. In the current study, we set out to reveal a novel transcriptional mechanism required for CD4 + T cell immunoactivity involved in SLE. First of all, miR-124 was experimentally determined to be under-expressed in peripheral blood samples of SLE patients relative to healthy individuals. We further isolated CD4 + T cells from the peripheral blood samples of SLE patients and healthy individuals, and found that miR-124 was poorly expressed in peripheral blood-derived CD4 + T cells of SLE patients. Subsequent experiments demonstrated that re-expression of miR-124 inhibited the immunoactivity of CD4 + T cells from SLE patients, which was achieved through the down-regulation of IRF1 since dual-luciferase reporter gene assay findings indicated that miR-124 could target IRF1. In addition, HDAC1 was found to be enriched at the miR-124 promoter resulting in inhibition of miR-124 expression, thereby promoting the immunoactivity of CD4 + T cells. In conclusion, we identify that as a stimulator of CD4 + T cell immunoactivity, HDAC1 may be implicated in the immunopathology of SLE. The study will open up new avenues to explore future immunotherapy strategies for SLE.

We previously reported that protein tyrosine phosphatase non-receptor type 3 (PTPN3), which is upregulated in activated lymphocytes, acts as an immune checkpoint. However, the mechanism by which PTPN3 expression is enhanced in activated lymphocytes is unknown. In this study, we analyzed the mechanism of PTPN3 expression in activated lymphocytes with a view for developing a novel immune checkpoint inhibitor that suppresses PTPN3. Through the activation process, lymphocytes showed enhanced NFκB activation as well as increased PTPN3 expression. NFκB enhanced proliferation, migration, and cytotoxicity of lymphocytes. Furthermore, NFκB enhanced PTPN3 expression and tyrosine kinase activation. TGFβ reduced PTPN3 expression and NFκB activation in the cancer microenvironment, and suppressed the biological activity of lymphocytes. The results of this study are expected to provide significant implications for improving existing immunotherapy and developing novel immunotherapy.

Germinal centers (GCs), which are the site of antibody diversification and affinity maturation, are vitally important for humoral immunity. GC B cell proliferation is essentially for these processes by providing enough templates for somatic hypermutation (SHM) and serving as a critical mechanism of positive selection. In the current study, we found a significant reduction of GC response in the spleens of GC B cell specific PHF14 knockout (PHF14GCB KO) mice compared with the wild-type control (PHF14GCB WT) when the mice were challenged with SRBCs or lymphocytic choriomeningitis virus. We also demonstrated that PHF14 did not affect the cell survival of GC B cells, but regulated the proliferation of GC B cells. In addition, PHF14 suppressed the expression of Cdkn1a (p21) though regulating the level of H3K4me3 to control the proliferation of GC B cells. Collectively, our data suggest that PHF14 plays an important role in the process of germinal center formation by regulating GC B cell proliferation in spleen.

Clonal anergy and depletion of antigen-specific CD8+ T cells are characteristics of immunosuppressed patients such as cancer and post-transplant patients. This has promoted translational research on the adoptive transfer of T cells to restore the antigen-specific cellular immunity in these patients. In the present work, we compared the capability of PBMCs and two types of mature monocyte-derived DCs (moDCs) to prime and to expand ex-vivo antigen-specific CD8+ T cells using culture conditioned media supplemented with IL-7, IL-15, and IL-21. The data obtained suggest that protocols involving moDCs are as efficient as PBMCs-based cultures in expanding antigen-specific CD8+ T cell to ELA and CMV model epitopes. These three gamma common chain cytokines promote the expansion of naive-like and central memory CD8+ T cells in PBMCs-based cultures and the expansion of effector memory T cells when moDCs were used. Our results provide new insights into the use of media supplemented with IL-7, IL-15, and IL-21 for the in-vitro expansion of early-differentiated antigen-specific CD8+ T cells for immunotherapy purposes.

Although hematopoietic stem cell transplantation (HSCT) has been widely used in the treatment of many diseases, graft-versus-host disease (GVHD) remains a major complication after allogeneic HSCT. Butyrophilin-like 2 (BTNL2) protein has been reported to have the ability to inhibit T cell proliferation in vitro; its ability to inhibit T cell responses in vivo has not been determined. We show here that in vivo administration of recombinant BTNL2-IgG2a Fc (rBTNL2-Ig) fusion protein ameliorates GVHD in mice. This is related to the ability of rBTNL2- Ig to inhibit T cell proliferation, activation and Th1/Th17 cytokine production in vivo. Furthermore, rBTNL2-Ig treatment increases the generation of regulatory T cells. Our results suggest that rBTNL2-Ig has the potential to be used in the prevention and treatment of patients with GVHD.

CD99, a leukocyte surface glycoprotein, has been implicated in many cellular processes including cell adhesion, cell migration and T cell activation. Our previous study demonstrated the anti-CD99 monoclonal antibody (mAb) clone MT99/3 inhibited T cell activation; however, the mechanism is unclear. In this study, we demonstrated that CD99 expressed on monocytes played a role in the inhibition of T cell activation. Anti-CD99 mAb MT99/3 downregulated the expression of costimulatory molecule CD86, but upregulated IL-6, IL-10 and TNF-α production by monocytes. The inhibitory effect of mAb MT99/3 required cell to cell contact between monocytes and lymphocytes. The soluble mediators produced by monocytes alone were insufficient to induce hypo-function of T lymphocytes. In summary, we demonstrated that ligation of CD99 on monocytes by anti-CD99 mAb MT99/3 could mediate T cell hypo-responsiveness. These findings provide the first evidence of the role of CD99 on monocytes that contributes to T cell activation.

CD8+ T cell differentiation is controlled by the transcription factors T-bet and Eomesodermin, in concert with the cytokines IL-2, IL-10 and IL-12. Among these pathways, the mechanisms by which T-box proteins and IL-10 interact to promote a memory T cell fate remain poorly understood. Here, we show that Eomes and IL-10 drive a central memory phenotype in murine CD8+ T cells. Eomes expression led to increased IL-10 expression by the effector CD8+ T cells themselves as well as an increase in the level of the lymph node homing selectin CD62L. Furthermore, exposure of effector CD8+ T cells to IL-10 maintained CD62L expression levels in culture. Thus, Eomes promotes a step-wise transition of effector T cells towards a memory phenotype, synergizing with IL-10 to enhance the expression of CD62L. The early augmentation of lymph node homing markers by Eomes may facilitate the retention of effector T cells in the relatively low inflammatory milieu of the secondary lymphoid organs that promotes central memory development.

Immune dysfunction can occur during sepsis or following major trauma. Decreased monocyte HLA-DR expression and cytokine responses are associated with mortality. Recent studies have shown that adaptive immune system defects can also occur in such patients, characterised by increased PD-L1 expression and associated T-cell anergy. The aim of this study was to determine the effects of an immune adjuvant, interferon-gamma, on monocyte PD-L1 expression and T-cell activation in an ex-vivo human whole blood model of infection. We found that with interferon-gamma treatment, monocytes had increased HLA-DR expression and augmented TNF-α production in response to LPS stimulation, with a decrease in IL-10 levels. Both LPS and interferon-gamma increased the level of monocyte PD-L1 expression, and that a combination of both agents synergistically stimulated a further increase in PD-L1 levels as measured by flow cytometry. However, despite elevated PD-L1 expression, both CD4 and CD8 T-cell activation was not diminished by the addition of interferon-gamma treatment. These findings suggest that PD-L1 may not be a reliable marker for T-cell anergy, and that interferon-gamma remains an adjuvant of interest that can improve the monocyte inflammatory response while preserving T-cell activation.

The CBM complex, which is composed of the proteins CARMA1, BCL10, and MALT1, serves multiple pivotal roles as a mediator of T-cell receptor and B-cell receptor-dependent NF-κB induction and lymphocyte activation. CARMA1, BCL10, and MALT1 are each proto-oncoproteins and dysregulation of CBM signaling, as a result of somatic gain-of-function mutation or chromosomal translocation, is a hallmark of multiple lymphoid malignancies including Activated B-cell Diffuse Large B-cell Lymphoma. Moreover, loss-of-function as well as gain-of-function germline mutations in CBM complex proteins have been associated with a range of immune dysregulation syndromes. A wealth of detailed structural information has become available over the past decade through meticulous interrogation of the interactions between CBM components. Here, we review key findings regarding the biochemical nature of these protein-protein interactions which have ultimately led the field to a sophisticated understanding of how these proteins assemble into high-order filamentous CBM complexes. To date, approaches to therapeutic inhibition of the CBM complex for the treatment of lymphoid malignancy and/or auto-immunity have focused on blocking MALT1 protease function. We also review key studies relating to the structural impact of MALT1 protease inhibitors on key protein-protein interactions.

Mutations causing loss of the NF-kappaB regulator IkappaBNS, result in impaired development of innate-like B cells and defective plasma cell (PC) differentiation. Since productive PC differentiation requires B cell metabolic reprogramming, we sought to investigate processes important for this transition using the bumble mouse strain, deficient for IkappaBNS. We report that LPS-activated bumble B cells exhibited elevated mTOR activation levels, mitochondrial accumulation, increased OXPHOS and mROS production, along with a reduced capacity for autophagy, compared to wildtype B cells. Overall, our results demonstrate that PC differentiation in the absence of IkappaBNS is characterized by excessive activation during early rounds of B cell division, increased mitochondrial metabolism and decreased autophagic capacity, thus improving our understanding of the role of IkappaBNS in PC differentiation.

ST6Gal1 is a critical sialyltransferase enzyme that controls the addition of α2,6-linked sialic acids to the termini of glycans. Attachment of sialic acids to glycoproteins as a posttranslational modification influences cellular responses, and is a well-known modifier of immune cell behavior. ST6Gal1 activity impacts processes such as: effector functions of immunoglobulin G via Fc sialylation, hematopoietic capacity by hematopoietic stem and progenitor cell surface sialylation, and lymphocyte activation thresholds though CD22 engagement and inhibition of galectins. This review summarizes recent studies that suggest α2,6 sialylation by ST6Gal1 has an immunoregulatory effect on immune reactions.

Platelets modulate the immune system following injury by interacting with CD4+ T regulatory cells (CD4+ Tregs). The underlying mechanisms remain unsolved. We hypothesize paracrine interactions via Tumor necrosis factor-alpha (TNFα)-, Toll like receptor-4 (TLR4)-, and Interleukin-10 (IL-10). In the murine burn injury model, CD4+ Treg activation pathways were selectively addressed using TNFR2-, TLR4- and IL-10-deficient mice. The CD4+ Treg signalling molecule PKC-θ was analyzed using phospho-flow cytometry to detect rapid cell activation. Thromboelastometry (ROTEM®) was used to assess platelet activation. Injury induced significant early activation of CD4+ Tregs, disruption of TNFR2 and TLR4 activation pathways resulted in lower activity. The disruption of IL-10 crosstalk had no significant impact. Selective disruption of paracrine interactions is associated with changes in posttraumatic hemostasis parameters. TNFR2- and TLR4-dependent pathways modulate the activation of CD4+ Tregs following trauma. In contrast, we did not observe a role of IL-10 in the posttraumatic activation of CD4+ Tregs. One sentence summary TLR4- and TNFR2-dependent mechanisms, but not IL-10-dependent pathways, modulate the anti-inflammatory response of CD4+ Tregs following trauma.

Immune active cells (IACs) have been shown to be an alternative immunotherapy for CHB patients. However, there is a practical problem of different expansion rate and function of HBV inhibition as individual variability exists. Our previous studies have confirmed that the proliferation and cytolysis of IACs were significantly up-regulated by engineered cells for costimulatory enhancement (ECCE) delivering a 4-1BBζ activating signal. In this study, we aimed to investigate the contribution of ECCE to IACs from CHB patients. We found that ECCE could enhance larger-scale expansion of IACs and the levels of HBV-markers were reduced prominently with minimal cytolysis, in the indirect system which separated ECCE-IACs and HepG2.2.15 by a 0.4-μm membrane. Furthermore, ECCE-IACs produced a lot of IFN-γ and TNF-α. Blockading them, the inhibition was abrogated. These results provide direct evidence that ECCE-IACs efficiently control HBV replication in a noncytolytic manner, and this effect is mediated by IFN-γ and TNF-α.

Restimulation-induced cell death (RICD) is an apoptotic program that regulates effector T cell expansion, triggered by repeated stimulation through the T cell receptor (TCR) in the presence of interleukin-2 (IL-2). Although CD4(+) regulatory T cells (Tregs) consume IL-2 and experience frequent TCR stimulation, they are highly resistant to RICD. Resistance in Tregs is dependent on the forkhead box P3 (FOXP3) transcription factor, although the mechanism remains unclear. T cells from patients with X-linked lymphoproliferative disease (XLP-1), that lack the adaptor molecule SLAM-associated protein (SAP), are also resistant to RICD. Here we demonstrate that normal Tregs express very low levels of SAP compared to conventional T cells. FOXP3 reduces SAP expression by directly binding to and repressing the SH2D1A (SAP) promoter. Indeed, ectopic SAP expression restores RICD sensitivity in human FOXP3(+) Tregs. Our findings illuminate the mechanism behind FOXP3-mediated RICD resistance in Tregs, providing new insight into their long-term persistence.

Autoimmune hepatitis (AIH) is a chronic autoimmune disease with the primary focus on autoreactive Th1-mediated response and cytotoxic T cells reaction. The contribution of natural killer cells (NK cells) to AIH remains poorly understood. In this project, we revealed that the frequency of peripheral NK cells, more accurately CD56dimNK subset, was reduced in AIH patients. The reduction of CD56dimNK was negatively correlated with disease progression. In experimental autoimmune hepatitis (EAH), hepatic accumulation of NK cells is observed along with a decrease of NK cells in the periphery. In addition, infiltrated NK cells are almost conventional CXCR3- NK cells, the counterpart of CD56dimNK cells in the human. Furthermore, conventional CXCR3-NK cells of infiltration and liver resident NK cells are activated progressively at active phase. Therefore, recruitment of cytotoxic NK cells into the liver, but not liver resident NK cells expansion, may partly account for AIH progression.

We investigated the direct effects of human granulocyte colony-stimulating factor (G-CSF) on functionality of human T-cell subsets. CD3+ T-lymphocytes were isolated from blood of healthy donors by positive magnetic separation. T cell activation with particles conjugated with antibodies (Abs) to human CD3, CD28 and CD2 molecules increased the proportion of cells expressing G-CSF receptor (G-CSFR, CD114) in all T cell subpopulations studied (CD45RA+/CD197+ naive T cells, CD45RA−/CD197+ central memory T cells, CD45RA−/CD197− effector memory T cells and CD45RA+/CD197− terminally differentiated effector T cells). Upon T-cell activation in vitro, G-CSF (10.0 ng/ml) significantly and specifically enhanced the proportion of CD114+ T cells in central memory CD4+ T cell compartment. A dilution series of G-CSF (range, 0.1–10.0 ng/ml) was tested, with no effect on the expression of CD25 (interleukin-2 receptor α-chain) on activated T cells. Meanwhile, G-CSF treatment enhanced the proportion of CD38+ T cells in CD4+ naive T cell, effector memory T cell and terminally differentiated effector T cell subsets, as well as in CD4− central memory T cells and terminally differentiated effector T cells. G-CSF did not affect IL-2 production by T cells; relatively low concentrations of G-CSF down-regulated INF-γ production, while high concentrations of this cytokine up-regulated IL-4 production in activated T cells. The data obtained suggests that G-CSF could play a significant role both in preventing the development of excessive and potentially damaging inflammatory reactivity, and in constraining the expansion of potentially cytodestructive T cells.

Tetraspanin membrane protein, epithelial membrane protein 3 (Emp3), is expressed in lymphoid tissues. Herein, we have examined the Emp3 in antigen presenting cell (APC) function in the CD8+ cytotoxic T lymphocytes (CTLs) induction. Emp3-overexpressing RAW264.7 macrophage cell line derived from BALB/c mice reduced anti-C57BL/6 alloreactive CTL induction, while Emp3-knockdown RAW264.7 enhanced it compared with parent RAW267.4. Emp3-overexpressing RAW264.7 inhibited, but Emp3-knockdown RAW264.7 augmented, CD8+ T cell proliferation, interferon-γ secretion, IL-2 consumption, and IL-2Rα expression on CD8+ T cells. The supernatant from co-culture with Emp3-overexpressing RAW264.7 contained higher amount of TNF-α, and TNF- α neutralization significantly restored all these inhibitions and the alloreactive CTL induction. These results suggest that Emp3 in allogeneic APCs possesses the inhibitory function of alloreactive CTL induction by downregulation of IL-2Rα expression CD8+ T cells via an increase in TNF-α production. This demonstrates a novel mechanism for regulating CTL induction by Emp3 in APCs through TNF-α production.

Cyclosporin A (CsA) is a well-known immunosuppressant that is used against steroid-resistant asthma. Group 2 innate lymphoid cells (ILC2s) and type 2 helper T (Th2) cells produce Th2 cytokines including IL-5 and play important roles in asthma pathogenesis. Here, we studied the effects of CsA in allergen-induced lung inflammation in mice and found that CsA decreased the number of lung ILC2s and attenuated papain-induced activation of ILC2s accompanied with IL-5 expression. The ILC2 suppression mediated by CsA was not observed in culture or in lymphocyte-deficient Rag2-/- mice. Thus, we propose a new suppressive effect of CsA, i.e., administration of CsA indirectly suppresses maintenance and activation of lung ILC2s in addition to direct suppression of T-cell activation and cytokine production.

Myeloid-derived suppressor cells (MDSCs) are immature myeloid cells critical in mediating immune suppression in cancer patients. To develop an in vitro assay system that functionally mimics the tumor microenvironment, we cultured human monocytes with conditioned media from several cancer cell lines. Conditioned media from five tumor cell lines induced survival and differentiation of monocytes into cells characteristically similar to macrophages and MDSCs. Notably, media from the 786.O renal cell carcinoma line induced monocytes to acquire a monocytic MDSC phenotype characterized by decreased HLA-DR expression, increased nitric oxide production, enhanced proliferation, and ability to suppress autologous CD3+ T cell proliferation. We further demonstrated that these in vitro MDSCs are phenotypically and functionally similar to patient-derived MDSCs. Inhibitors of STAT3, CK2, and GM-CSF resulted in partial reversal of the MDSC phenotype. MDSCs generated in vitro from 786.O tumor conditioned media represent a platform to identify potential therapeutics that inhibit MDSC activities.

Human regulatory dendritic cells (DCreg) were generated from CD14 immunobead-purified or elutriated monocytes in the presence of vitamin D3 and IL-10. They exhibited similar, low levels of costimulatory CD80 and CD86, but comparatively high levels of co-inhibitory programed death ligand-1 (PD-L1) and IL-10 production compared to control immature DC (iDC). Following Toll-like receptor 4 ligation, unlike control iDC, DCreg resisted phenotypic and functional maturation and further upregulated PD-L1:CD86 expression. Whereas LPS-stimulated control iDC (mature DC; matDC) secreted pro-inflammatory tumor necrosis factor but no IL-10, the converse was observed for LPS-stimulated DCreg. DCreg weakly stimulated naive and memory allogeneic CD4+ and CD8+ T cell proliferation and IFNγ, IL-17A and perforin/granzyme B production in MLR. Their stimulatory function was enhanced however, by blocking PD-1 ligation. High-throughput T cell receptor (TCR) sequencing revealed that, among circulating T cell subsets, memory CD8+ T cells contained the most alloreactive TCR clonotypes and that, while matDC expanded these alloreactive memory CD8 TCR clonotypes, DCreg induced more attenuated responses. These findings demonstrate the feasibility of generating highly-purified GMP-grade DCreg for systemic infusion, their influence on the alloreactive T cell response, and a key mechanistic role of the PD1 pathway.

T lymphocytes play crucial roles in adaptive immune responses to tumors. However, due to different tolerance mechanisms and inhibitory effects of the tumor microenvironment (TME) on T cells, responses to tumors are insufficient. In fact, cellular and molecular suppressive mechanisms repress T cell responses in the TME, resulting in senescent, anergic and exhausted lymphocytes. Exhaustion is a poor responsive status of T cells, with up-regulated expression of inhibitory receptors, decreased production of effective cytokines, and reduced cytotoxic activity. Low immunogenicity of tumor antigens and inadequate presentation of tumor-specific antigens results in inappropriate activation of naive T lymphocytes against tumor antigens. Moreover, when effector cytotoxic T cells enter TME, they encounter a complicated network of cells and cytokines that suppress their effectiveness and turn them into exhausted T cells. Thus, the mechanism of T cell exhaustion in cancer is different from that in chronic infections. In this review we will discuss the main components such as inhibitory receptors, inflammatory cells, stromal cells, cytokine milieu as well as environmental and metabolic conditions in TME which play role in development of exhaustion. Furthermore, recent therapeutic methods available to overcome exhaustion will be discussed.

Lung cancer cells harboring multiple mutations as a consequence of long-term damage by different etiologic factors are responsible for high immunogenicity. Immune checkpoint inhibitors significantly improve treatment results in non-small cell lung cancer (NSCLC). Unfortunately, the role of T-lymphocytes in early NSCLC has not been sufficiently elucidated. The aim of this study was to characterize peripheral blood T cells expressing several selected surface antigens (CD4, CD8, CD25, CD28, PD-1, CTLA-4) and transcription factors (T-bet, ROR-yt, Fox-P3, GATA-3) in this patient population. The study group (LC) consisted of 80 treatment-naïve patients with T1/2aN0M0 NSCLC and was compared with 40 cancer-free patients matched for non-oncological diseases and demographic parameters (CG). Significantly higher counts of CTLA-4+cells (in both CD4+and CD8+subtypes), a lower proportion of PD-1 expressing cells and a significantly higher percentage of Fox-P3+CD4+cells were found in the LC group. The high proportion of CD4+PD-1+cells significantly correlated with poor outcomes in LC group, while low CD4/CD8 ratio predicted a better prognosis. Based on our results it seems that NSCLC even at early stages of development initiate changes in the proportions of T cells that may have a significant impact on the clinical outcome.

Severe aplastic anemia (SAA) is an autoimmune disease characterized by severe pancytopenia and bone marrow failure. In our previous studies, we found natural killer (NK) cells were aberrant in SAA patients. T cell immunoglobulin mucin-3 (TIM-3), an important regulator of immunity, is widely detected on NK cells and may contribute as a marker of activation and maturation of NK cells. In this study, we found that SAA untreated patients had lower TIM-3 expression on NK cells and CD56dim NK subsets compared with normal controls, and were correlated with the severity of pancytopenia of SAA. After immunosuppressive therapy (IST), TIM-3 expression recovered to normal level. Moreover, the TIM-3 mRNA levels in NK cells significantly increased in SAA remission patients after IST. We inferred that low expression of TIM-3 on NK cells might lead to NK cells dysfunction and involve in the progress of bone marrow failure in SAA.

Parasite infections in the developing world have been considered to promote resistance to immune-mediated diseases such as asthma. Mouse studies have shown that helminths and their products reduce the development of allergic asthma. Since epidemiologic studies that show similar protection are in relation to geohelminth infections that occur in early life, we hypothesized that the parasite-mediated protection against asthma may differ by age. Mice infected with Heligmosomoides polygyrus at 3-weeks of age had similar asthma phenotype compared to mice infected at 28-weeks of age wherein airway eosinophilia was unaltered but tissue inflammation and GC metaplasia were reduced. In contrast, mice infected at 18-weeks of age had elevated macrophagic airway inflammation with accompanying tissue pathology. The presence of γδ T cells and Treg cells in the airways was also regulated by age at worm infection. Our findings demonstrate the importance of age in immune responses that may regulate gut and lung diseases.

Common variable immunodeficiency (CVID) is a heterogeneous group of primary antibody deficiencies (PAD) which is defined by recurrent infections, hypogammaglobulinemia and defects in B-cell differentiation into plasma cells and memory B cells. T cell abnormalities have also been described in CVID patients. Several studies reported that Treg frequencies and their functional characteristics are disturbed and might account for the aberrant immune responses observed in CVID patients. The aim of this review is to describe phenotypic and functional characteristics of Treg cells, and to review the literature with respect to the reported Treg defects and its association with the clinical manifestation in CVID.

Allergen-induced airway inflammation is characterized by Th2-mediated eosinophilic inflammation in the lungs. While the molecular mechanisms leading to this abnormal Th2 response remain unclear. Recent studies have demonstrated that MicroRNAs (miRNAs) modulate allergic airway inflammation. In this study, the role of miRNAs in allergic asthma pathogenesis was examined. Differentially expressed miRNAs were identified via miRNA microarray, with miR-155 being among the most highly expressed in asthma mice lungs. Examination of miR-155 overexpression resulted in enhanced inflammation and mucus hypersecretion in the lungs of allergen-challenged mice compared with control animals. Furthermore, CTLA-4, an important negative regulator of T-cell activation, was identified as a direct miR-155 target. Moreover, miR-155 overexpression in CD4+ T cells resulted in decreased CTLA-4 levels and a subsequent increased proliferative response. Collectively, these findings suggest that miR-155 might contribute to allergic asthma by increasing the proliferative response of Th cells via CTLA-4 downregulation and thus may be a potential therapeutic target for allergic asthma.

Co-relation between allergic asthma and microbiota varying with diet has been extensively investigated, implicating that oral supplement of alternative pharmaceuticals are potential for asthma control. Probiotics are of great concern due to its beneficial effects on the host, whereas the potential mechanisms and the optimal dose need to be further explored. In the present study, three different doses of mixed strains were given orally to mouse model of allergic asthma induced by ovalbumin (OVA). Continuous administration of mixed probiotics could alleviate OVA-induced allergic inflammation through reducing costimulatory molecules on the surface of dendritic cells (DCs) and increasing mucosal gut-primed Tregs induced by mesenteric CD103+DCs. Mixed probiotics-induced protective effect relates to gut microbiota instead of lung microbes. Microbial diversity and Bacteroidetes/Firmicutes (B/F) ratio are augmented upon probiotic strains. Interestingly, treatment with mixed strain lead to an increased levels of genus Lactobacillus. Lactobacillus genus-Operational Taxonomic Unit (OUT) analysis indicated that OTU9 may be the dominant strain from mixed probiotics providing protective effect. The low dose seems to be the best in the whole study. Our results provide new evidence for probiotic application in allergic diseases and support the idea that targeted gut microbiota will be an effective approach for allergic airway diseases.

The in vivo proliferation and viability of transfused engineered T cells markedly limits the long-term effect of adoptive cell therapy on tumors. The therapeutic efficacy and proliferative potential of T cells are reported to be dependent on the differentiation status of T cells. The T cells at the early stage of progressive differentiation have a long lifespan, strong proliferative potential, and the ability to reconstruct intact T cell subsets. Thus, they are more suitable for adoptive immunotherapy. Previously, it was difficult to obtain a sufficient number of early differentiated T cells by inhibiting the progressive differentiation of T cells or by two-step programming. A more effective strategy is to directly reprogram and dedifferentiate the easily available terminal effector T (TEFF) cells, which are generated in large numbers, into early T cells. This study attempted to overexpress eight (candidate) early differentiation-specific transcription factors (TFs) (LEF1, KLF7, ID3, EOMES, BCL6, TCF7, FOXP1, and FOXO1) in the TEFF cells, which were activated by in vitro stimulation, to promote dedifferentiation into early T cells. In the mature TEFF cells simultaneously overexpressing these specific TFs, the expression pattern of T cell differentiation markers (CCR7 and CD45RO) exhibited a tendency to change to the pattern observed during early differentiation. The transcriptome analysis revealed that the function of differentially expressed genes was mainly concentrated in the cell cycle, growth and development, and effector function. Moreover, many genes related to early differentiated T cells (such as BCL2 and PIM1) were significantly upregulated, while those related to the effector function of TEFF cells were significantly downregulated (such as GZMB, PRF1, and GNLY). Additionally, the TEFF cells overexpressing characteristic TFs exhibited enhanced anti-apoptotic capabilities and decreased secretion of cytokines (IFN-γ and TNF-α). Based on these results, we believe that the TEFF cells were reprogrammed into a less differentiated state after overexpression of the eight specific TFs.

NFAT2 activity was shown to be of critical importance in B cell receptor signaling, development and proliferation; however its role in B cell development in the periphery is still not completely understood. We confirmed that NFAT2 deletion leads to impaired B1 B cell development, supported by our finding of limited B1 progenitors in the bone marrow and spleen of NFAT2 deficient mice. Moreover, we show for the first time that loss of NFAT2 increases immature B cells in particular transitional T2 and T3 as well as mature follicular B cells while marginal zone B cells are decreased. We further demonstrate that NFAT2 regulates the expression of B220, CD23, CD38, IgM/IgD and ZAP70 in murine B cells. In vivo analyses revealed decreased proliferation and increased apoptosis of NFAT2 deficient B cells. In summary, this study provides an extensive analysis of the role of NFAT2 in peripheral B lymphocyte development.

Pathogenic microorganisms utilize multiple approaches to break down host immunity in favor of their invasion, of which, cystatin C is one of the soluble factors secreted by parasites reported to affect host immunity in vivo. The cellular targets and mechanisms of action in vivo of cystatin C, however, are far from clear. As professional antigen-presenting cells, dendritic cells (DCs) are first immune cells that contact foreign pathogenic agents or their products to initiate immune responses. We previously reported that cystatin C can regulate the functions of DCs in terms of suppressed CD4+ T cell activation but enhanced Th1/Th17 differentiation via different mechanisms. Here, we further verified these regulatory effects of cystatin C on DCs in vivo. We found that the suppressive role of DC-mediated CD4+ T cell proliferation by cystatin C was partly cell-contact independent and extended to CD8+ T cells in vivo. Although cystatin C-overexpressing DCs trafficked equally as their mock-transduced counterparts, their adoptive transfer suppressed CD8+ T cell immunity and resulted in compromised tumor rejection in both vaccination and treatment regimes. Compared with their role in promoting Th17 differentiation in vivo, cystatin C-transduced DCs had far greater ability to induce T regulatory cells (Tregs), leading to collectively a higher Treg/Th17 ratio in an adoptively transferred disease model, and thus relieved Th17-dependent autoimmunity. Collectively, these data demonstrated strong in vivo evidences for immune regulatory roles of cystatin C in DCs and provided theoretical basis for the application of cystatin C-transduced cell therapy in the treatment or remission of certain autoimmune diseases. (246).

Cytotoxic T lymphocytes (CTLs) play a major role in cancer immunotherapy. A potent tumor immunotherapy may not only require activation of anti-tumor effector cells but also rely on the use of cytokines to create a controlled environment for the development of anti-tumor T cells. In this study, we fabricated a dual-target immunonanoparticle, e.g. poly(d,l-lactide-co-glycolide) nanoparticle, by loading Interleukin-12 (IL-12) and modifying with CD8 and Glypican-3 antibodies on the surface. Our results demonstrate that the fabricated targeting immunonanoparticles bind specifically to the two target cells of interest, i.e. CD8+ T cells and HepG-2 cells via the antibody-antigen interactions and form T cell-HepG-2 cell clusters, which enhances the cytotoxicity of T cells. IL-12-containing dual-target immunonanoparticles delivered IL-12 specifically to CD8+ T cells, and favored the expansion, activation and cytotoxic activity of CD8+ T lymphocytes. These results suggest that dual-target IL-12-encapsulated nanoparticles are a promising platform for cancer immunotherapy.

The clonal proliferation of antigen-specific T cells during an immune response critically depends on the differential response to growth factors, such as IL-2. While activated T cells proliferate robustly in response to IL-2 stimulation, naive (quiescent) T cells are able to ignore the potent effects of growth factors because they possess chromatin that is tightly condensed such that transcription factors, such as STAT5, cannot access DNA. Activation via the T cell receptor (TCR) induces a rapid decondensation of chromatin, permitting STAT5-DNA engagement and ultimately promoting proliferation of only antigen-specific T cells. Previous work demonstrated that the mobilization of intracellular calcium following TCR stimulation is a key event in the decondensation of chromatin. Here we examine PKC-dependent signaling mechanisms to determine their role in activation-induced chromatin decondensation and the subsequent acquisition of competence to respond to IL-2 stimulation. We found that a calcium-dependent PKC contributes to activation-induced chromatin decondensation and that the p38 MAPK and NFκB pathways downstream of PKC each contribute to regulating the proper decondensation of chromatin. Importantly, we found that p44/42 MAPK activity is required for peripheral T cells to gain competence to properly respond to IL-2 stimulation. Our findings shed light on the mechanisms that control the clonal proliferation of antigen-specific peripheral T cells during an immune response.

Chronic infection by Trypanosoma cruzi decreases T cell proliferation and it is most likely accompanied by changes in signals required for activation. We assessed the effect of T. cruzi antigens on mitogen-induced proliferation of T cells from uninfected individuals and the association with the expression of molecules involved in antigen presentation, T cell costimulation and activation, and cytokine production. T. cruzi antigen exposure reduced mitogen-induced proliferation of CD4+ and CD8+ T cells in PBMC cultures, but only reduced mitogen-induced proliferation in the CD4+ T cells from sorted cell cultures cocultured with antigen-pulsed CD3− cells. CD40/CD80 and CD86 expression were reduced in antigen-pulsed DCs and monocytes, respectively. TNF-α, IL-10 and CCL17 levels were increased in cultures with antigen-pulsed CD3− cells, while CD3ζ chain expression was reduced in T cells from cultures with antigen. Our findings suggest that T. cruzi could alter T cell proliferation indirectly by downregulating costimulatory molecules and inducing the secretion of IL-10 and directly by decreasing TCR signaling.

Many aspects remain elusive of the mechanisms governing T cell quiescence. Here we show that E protein activity helps to establish a quiescent program in naïve T cells. Decreased E protein activity, as the consequence of enforced expression of an Id1 transgene, led to the accumulation of CD4

Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of immature myeloid cells that expand in inflammatory conditions including transplantation. MDSCs may be capable of controlling rejection. The critical mechanisms underlying MDSC mediated alloregulation remain unexplored. G-CSF potently stimulates MDSC expansion. We hypothesized that G-CSF-induced MDSCs use a novel mechanism to suppress T cell responses. G-CSF promoted expansion of MDSCs and enhanced their suppressive function against T cell proliferation. Gene expression analysis revealed MDSCs expanded with G-CSF upregulated immune-related genes, but downregulated proliferation-related genes when compared to naïve control MDSCs. The KIT oncogene, encoding the c-Kit (CD117) transmembrane tyrosine kinase receptor, was the most significantly increased in MDSCs expanded with G-CSF. c-Kit inhibition with both imatinib and monoclonal blocking antibody reduced expression of ARG-1, iNOS, PD-L1, and SAA3. Further, imatinib also reduced MDSC-mediated T cell suppression in vitro. Modulation of c-Kit activity may represent a therapeutic target for alloregulatory MDSCs.