Single-cell analysis can define distinct evolution of tumor sites in follicular lymphoma.

Tumor heterogeneity complicates biomarker development and fosters drug resistance in solid malignancies. In lymphoma, our knowledge of site-to-site heterogeneity and its clinical implications is still limited. Here, we profiled 2 nodal, synchronously acquired tumor samples from 10 patients with follicular lymphoma (FL) using single-cell RNA, B-cell receptor (BCR) and T-cell receptor sequencing, and flow cytometry. By following the rapidly mutating tumor immunoglobulin genes, we discovered that BCR subclones were shared between the 2 tumor sites in some patients, but in many patients, the disease had evolved separately with limited tumor cell migration between the sites. Patients exhibiting divergent BCR evolution also exhibited divergent tumor gene-expression and cell-surface protein profiles. While the overall composition of the tumor microenvironment did not differ significantly between sites, we did detect a specific correlation between site-to-site tumor heterogeneity and T follicular helper (Tfh) cell abundance. We further observed enrichment of particular ligand-receptor pairs between tumor and Tfh cells, including CD40 and CD40LG, and a significant correlation between tumor CD40 expression and Tfh proliferation. Our study may explain discordant responses to systemic therapies, underscores the difficulty of capturing a patient's disease with a single biopsy, and furthers our understanding of tumor-immune networks in FL.

The IL-33/ST2 pathway suppresses murine colon cancer growth and metastasis by upregulating CD40 L signaling.

Interleukin (IL)-33 is a member of the IL-1 family, participating in both helper T1 (Th1)- and Th2-type immune responses, but its ambiguous effects on tumor growth and related immune mechanisms remain unclear. Here, we report that recombinant mouse IL-33 (mIL-33) significantly inhibited colon cancer growth and metastasis to lung and liver in a murine CT26 or MC38 tumor-cell engraftment model. This effect could be associated with CD4+ T cells and CD40 L signaling, as depletion of CD4+ T cells or blocking CD40 L signaling in vivo partly abolished the antitumor function of IL-33. In addition, IL-33 treatment upregulated CD40 L expression on tumor-infiltrating lymphocytes, and promoted the activation of CD4+ T, CD8+ T and natural killer cells via CD40 L signaling. Furthermore, IL-33 was sufficient to induce the ST2 expression on CD4+ T cells, but not on CD8+ T and natural killer cells, indicating that IL-33 acted on CD4+ T cells via a positive-feedback loop. Our findings shed new light on the IL-33-mediated antitumor effects and mechanisms of Th1 action, and also suggest that IL-33 may serve as an activator to boost anticancer immune responses in singular or combinatory therapies.

Copy number of the X-linked genes TLR7 and CD40L influences innate and adaptive immune responses.

The number of the X chromosome-linked genes has been previously suggested to influence immune responses and the development of autoimmune diseases. In the present study, we aimed at evaluating the level of expression of CD40L (an X-linked gene involved in adaptive immunity) and TLR7 (an X-linked gene involved in innate immunity) in a variety of different karyotypes. Those included males, females and patients with X chromosome aneuploidy. Healthy females (46, XX; n = 10) and healthy males (46, XY; n = 10) were compared to females with Turner syndrome (TS) (45, X; n = 11) and males with Klinefelter syndrome (KS) (47, XXY; n = 5). Stimulation of peripheral blood mononuclear cells (PBMCs) with PMA and ionomycin resulted in higher percentage of CD3 + CD40L+ T cells (P < 0.001) and higher level expression of CD40L in T cell (P < 0.001) in female and KS patients compared with male and TS patients. TLR7-mediated IFN-alpha production by HLADR + CD3- CD19- cells was significantly upregulated in healthy women compared with healthy males, TS and KS patients (P < 0.001). TLR7 agonist-stimulated PBMCs from healthy females and KS patients expressed significantly higher levels of TLR7 mRNA than those from male and TS patients (P < 0.05). The increased expression of the X-linked genes TLR7 and CD40L in healthy females and KS patients suggests that the presence of two X chromosomes plays a major role in enhancing both innate and adaptive immune responses. These results may contribute to the explanation of sex-based differences in immune biology and the sex bias in predisposition to autoimmune diseases.

Combined therapy of infusion of DC from rats with higher expression of IDO and CD40L on rejection post heart transplantation.

OBJECTIVE: Indoleamine 2, 3-dioxygenase (IDO) can inhibit rejection of graft via inducing T cell apoptosis. CD40L monoclonal antibody (mAb) inhibits T cell activation. However, the effects of the combination of infusion of dendritic cell (DC) from IDO over-expressed donor mice and CD40L mAb on the treatment of graft rejection after heart transplantation have not been reported. MATERIALS AND METHODS: Allogeneic heart transplantation mouse model was established. Recipient mice were divided into three groups, including control group, IDO group (in which DC donors received adenoviral vector of IDO) and combined therapy group (which received both IDO over-expressed DC infusion and CD40L mAb injection post transplantation). Survival time and cardiac function were observed, with IDO expression being quantified. Flow cytometry (FCM) was used to analyze T cell apoptosis, while enzyme linked immunosorbent assay (ELISA) was adopted to test the levels of interferon-γ (IFN-γ), interleukin-2 (IL-2), interleukin-10 (IL-10) and interleukin-6 (IL-6). RESULTS: IDO expression was significantly elevated in both IDO and combined therapy groups, with enhanced T cell apoptosis compared to control group (p < 0.05). Both groups had better survival time and cardiac functions compared to control group, along with increased IL-10/IL-6 expression and suppressed INF-γ and IL-2 expression (p < 0.05). However, combined therapy had a better efficiency compared to IDO group (p < 0.05). CONCLUSIONS: Combined therapy of high IDO expressed mouse DC perfusion with CD40L mAb can elongate the survival time of recipient heart and inhibit rejection reaction via facilitating T cell apoptosis. Meanwhile, combined therapy could also regulate the expression of some immune suppressant factors and mediate the Th1/Th2 cytokine balance.

Increased expression of the membrane-bound CD40 ligand on peripheral CD4+ T cells in the acute phase of AQP4-IgG-seropositive neuromyelitis optica spectrum disorders.

Currently, no data are available regarding the expression levels of CD40L on CD4+ T cells in patients with neuromyelitis optica spectrum disorders (NMOSD). The percentage of circulating CD40L+CD4+ T cells was measured by flow cytometry in 23 NMOSD patients and 10 healthy controls. The ratio of CD40L+CD4+ to CD4+ T cells in patients at acute phase (18.28 ±â€¯15.56%) was significantly higher than that in healthy controls (7.23 ±â€¯5.94%, P = .032) and was positively correlated with disease severity (r = 0.532, P = .041). Thus, our results suggest an important role of this molecule in acute attacks of NMOSD.

Assessment of soluble platelet CD40L and CD62P during the preparation process and the storage of apheresis platelet concentrates: Absence of factors related to donors and donations.

Platelet transfusions may be associated with certain adverse effects in recipients, potentially caused by the presence of biological response modifiers contained in the platelet concentrates. The aim of this study is to identify the parameters that reflect platelet activation during both the preparation process and the storage of platelet concentrates. A total of 3,949apheresis platelet concentrate samples were studied with regard to parameters related to the donor as well as to the preparation process and their storage. Key glycoproteins characteristic of platelet activation, i.e. soluble CD40L and CD62P, were quantified in platelet concentrate supernatants on completion of their processing and during storage, using Luminex technology. We observed an increase in soluble factors over time. However, the different parameters studied in connection either with the donors or with the donations, such as (i) donor gender, (ii) donor blood group, (iii) time of collection and (iv) type of apheresis separator, do not seem to have any effect on platelet activation or the release of soluble CD40L and CD62P.

Activation and Induction of Antigen-Specific T Follicular Helper Cells Play a Critical Role in Live-Attenuated Influenza Vaccine-Induced Human Mucosal Anti-influenza Antibody Response.

There is increasing interest recently in developing intranasal vaccines against respiratory tract infections. The antibody response is critical for vaccine-induced protection, and T follicular helper cells (TFH) are considered important for mediating the antibody response. Most data supporting the role for TFH in the antibody response are from animal studies, and direct evidence from humans is limited, apart from the presence of TFH-like cells in blood. We studied the activation and induction of TFH and their role in the anti-influenza antibody response induced by a live-attenuated influenza vaccine (LAIV) in human nasopharynx-associated lymphoid tissue (NALT). TFH activation in adenotonsillar tissues was analyzed by flow cytometry, and anti-hemagglutinin (anti-HA) antibodies were examined following LAIV stimulation of tonsillar mononuclear cells (MNC). Induction of antigen-specific TFH by LAIV was studied by flow cytometry analysis of induced TFH and CD154 expression. LAIV induced TFH proliferation, which correlated with anti-HA antibody production, and TFH were shown to be critical for the antibody response. Induction of TFH from naive T cells by LAIV was shown in newly induced TFH expressing BCL6 and CD21, followed by the detection of anti-HA antibodies. Antigen specificity of LAIV-induced TFH was demonstrated by expression of the antigen-specific T cell activation marker CD154 upon challenge by H1N1 virus antigen or HA. LAIV-induced TFH differentiation was inhibited by BCL6, interleukin-21 (IL-21), ICOS, and CD40 signaling blocking, and that diminished anti-HA antibody production. In conclusion, we demonstrated the induction by LAIV of antigen-specific TFH in human NALT that provide critical support for the anti-influenza antibody response. Promoting antigen-specific TFH in NALT by use of intranasal vaccines may provide an effective vaccination strategy against respiratory infections in humans.IMPORTANCE Airway infections, such as influenza, are common in humans. Intranasal vaccination has been considered a biologically relevant and effective way of immunization against airway infection. The vaccine-induced antibody response is crucial for protection against infection. Recent data from animal studies suggest that one type of T cells, TFH, are important for the antibody response. However, data on whether TFH-mediated help for antibody production operates in humans are limited due to the lack of access to human immune tissue containing TFH In this study, we demonstrate the induction of TFH in human immune tissue, providing critical support for the anti-influenza antibody response, by use of an intranasal influenza vaccine. Our findings provide direct evidence that TFH play a critical role in vaccine-induced immunity in humans and suggest a novel strategy for promoting such cells by use of intranasal vaccines against respiratory infections.

Neutralization of CD40 ligand costimulation promotes bone formation and accretion of vertebral bone mass in mice.

Objective: Immunosuppressive biologics are used in the management of RA and additional immunomodulators are under investigation including modulators of the CD40/CD40 ligand (CD40L) costimulation pathway. Tampering with immune function can have unanticipated skeletal consequences due to disruption of the immuno-skeletal interface, a nexus of shared cells and cytokine effectors serving discrete functions in both immune and skeletal systems. In this study, we examined the effect of MR1, a CD40L neutralizing antibody, on physiological bone remodelling in healthy mice. Methods: Female C57BL6 mice were treated with MR1 and BMD was quantified by dual energy X-ray absorptiometry and indices of trabecular bone structure were quantified by micro-CT. Serum biochemical markers were used to evaluate bone turnover and formation indices by histomorphometry. Results: Unexpectedly, MR1 stimulated significant accretion of BMD and trabecular bone mass in the spine, but not in long bones. Surprisingly, bone accretion was accompanied by a significant increase in bone formation, rather than suppression of bone resorption. Mechanistically, MR1-induced bone accrual was associated with increased Treg development and elevated production of cytotoxic T lymphocyte antigen 4, a costimulation inhibitor that promotes T cell anergy and CD8+ T cell expression of the bone anabolic ligand Wnt-10b. Conclusion: Our studies reveal an unexpected bone anabolic activity of pharmacological CD40L suppression. Therapeutic targeting of the CD40L pathway may indeed have unforeseen consequences for the skeleton, but may also constitute a novel strategy to promote bone formation to ameliorate osteoporotic bone loss and reduce fracture risk in the axial skeleton.

Human CD40 ligand-expressing type 3 innate lymphoid cells induce IL-10-producing immature transitional regulatory B cells.

BACKGROUND: Type 3 innate lymphoid cells (ILC3s) are involved in maintenance of mucosal homeostasis; however, their role in immunoregulation has been unknown. Immature transitional regulatory B (itBreg) cells are innate-like B cells with immunosuppressive properties, and the in vivo mechanisms by which they are induced have not been fully clarified. OBJECTIVE: We aimed to investigate the ILC3-B-cell interaction that probably takes place in human tonsils. METHODS: ILC3s were isolated from peripheral blood and palatine tonsils, expanded, and cocultured with naive B cells. Tonsillar ILC3s and regulatory B cells were visualized with immunofluorescence histology. ILC3 frequencies were measured in tonsil tissue of allergic and nonallergic patients and in peripheral blood of allergic asthmatic patients and healthy control subjects. RESULTS: A mutually beneficial relationship was revealed between ILC3s and B cells: ILC3s induced IL-15 production in B cells through B cell-activating factor receptor, whereas IL-15, a potent growth factor for ILC3s, induced CD40 ligand (CD40L) expression on circulating and tonsillar ILC3s. IL-15-activated CD40L+ ILC3s helped B-cell survival, proliferation, and differentiation of IL-10-secreting, PD-L1-expressing functional itBreg cells in a CD40L- and B cell-activating factor receptor-dependent manner. ILC3s and regulatory B cells were in close connection with each other in palatine tonsils. ILC3 frequency was reduced in tonsil tissue of allergic patients and in peripheral blood of allergic asthmatic patients. CONCLUSION: Human CD40L+ ILC3s provide innate B-cell help and are involved in an innate immunoregulatory mechanism through induction of itBreg cell differentiation, which takes place in palatine tonsils in vivo. This mechanism, which can contribute to maintenance of immune tolerance, becomes insufficient in allergic diseases.

Blood Mixing Upregulates Platelet Membrane-Bound CD40 Ligand Expression In Vitro Independent of Abo Compatibility.

Platelets play a central role in the inflammation response via CD40 ligand (CD40L) expression, which may lead to transfusion reactions. The precise role of platelet CD40L-mediated inflammation in transfusion reactions is unclear. Therefore, we assessed the effects of in vitro blood mixing on platelet CD40L expression. In addition, we examined the effect of ABO compatibility on CD40L expression. Donor-packed red blood cells were acquired from a blood bank, and recipient blood was obtained from patients undergoing cardiac surgery and prepared as washed platelets. Donor blood was mixed with suspended, washed recipient platelets to obtain a final mixing ratio of 1%, 5%, or 10% (vol/vol). The blood mixtures were divided into three groups: group M, cross-matched blood-type mixing (n = 20); group S, ABO type-specific uncross-matched blood (n = 20); and group I, ABO incompatibility (not ABO type-specific blood and not process cross-matched) mixing (n = 20). The blood mixtures were used to detect platelet membrane-bound CD40L expression by flow cytometry. Blood mixing resulted in an increase in CD40L expression in group M (P < 0.001), group S (P < 0.001), and group I (P < 0.001). CD40L expression after blood mixing potentially led to a transfusion reaction in each of the groups. There were no differences in CD40L expression among the three groups (P = 0.988) correlated with ABO compatibility or incompatibility. This indicates that the reactions between red blood cell surface antigens and plasma antibodies do not play a role in the induction of CD40L expression.

Clinical significance of costimulatory molecules CD40/CD40L and CD134/CD134L in coronary heart disease: A case-control study.

The aim of the study was to evaluate the potential role of CD40/CD40 ligand (CD40L) and CD134/CD134 ligand (CD134L) in the development of coronary heart disease (CHD) via the performance of a case-control study.The research objects were 234 cases of CHD patients and 120 cases of well-matched normal controls. Following the separation of peripheral blood mononuclear cells (PBMCs), real-time quantitative PCR (qRT-PCR), Western blot, immunohistochemistry, and flow cytometry were applied for the detection of mRNA levels and expression levels of CD40/CD40L and CD134/CD134L; meanwhile, intercellular adhesion molecule-1 (ICAM-1) and Fas protein mRNA levels were detected using qRT-PCR.There was no statistical difference in the comparison of baseline characteristics between groups, indicating comparability between groups. qRT-PCR and Western blot analysis indicated that CD40/CD40L and CD134/CD134L mRNA and protein expression levels were all increased in the CHD group than those in the control group. Flow cytometry further confirmed the similar tendency. Meanwhile, ICAM-1 and Fas protein mRNA levels were elevated in the CHD group and positively correlated with the above parameters. Furthermore, CD40/CD40L expression rates were negatively correlated with gender and different types of CHD. Meanwhile, CD134/CD134L expressions were also higher in male patients, in patients with family history, previous history of hypertension, diabetes, and cerebrovascular diseases.CD40/CD40L and CD134/CD134L are increased and may have potential correlation with clinical pathological features of patients with CHD. Further in-depth exploration of costimulatory molecules for CHD guidance as well as intrinsic mechanisms are needed combined with in vivo and in vitro experiments.

CD40 Ligand and GMCSF Coexpression Enhance the Immune Responses and Protective Efficacy of PCV2 Adenovirus Vaccine.

Porcine circovirus 2 (PCV2) capsid protein (Cap) is the major structural protein that is responsible for neutralizing antibodies development and protective immunity, thus it is usually used to develop vaccines against porcine circovirus-associated disease (PCVAD). Porcine CD40 ligand (CD40L) and granulocyte-macrophage colony-stimulating factor (GMCSF) have positive immunostimulatory effects on immunocytes and have been applied in vaccine efficacy improvement as attractive adjuvant cytokines, respectively. However, whether these two cytokines can produce synergistic effect in vaccines still need to be further studied. In this study, porcine CD40L and GMCSF were inserted into recombinant adenoviruses to test the immunogenicity of PCV2 adenovirus vaccine in mice. Western blot and indirect immunofluorescence assay showed that Ad-Cap, Ad-CD40L-Cap, Ad-Cap-GMCSF, and Ad-CD40L-Cap-GMCSF were successfully constructed. Indirect ELISA and virus neutralizing assay showed that CD40L and GMCSF could enhance humoral immune responses, and PCV2 Cap-specific antibody titer and neutralizing activities were significantly higher in Ad-CD40L-Cap-GMCSF group than that in the other groups that just inserted either porcine CD40L or GMCSF in recombinant adenoviruses. Moreover, lymphocyte proliferation assay and cytokine release assay showed that CD40L and GMCSF enhanced the cellular immune responses of Ad-Cap, and had synergistic effects in lymphocyte proliferative activities and Th1-type cytokine production. Following PCV2 challenge, the viral loads in lungs of Ad-CD40L-Cap-GMCSF group were significantly lower compared with Ad-Cap, Ad-CD40L-Cap, and Ad-Cap-GMCSF group. Taken together, the results of this study demonstrated that CD40L and GMCSF could synergistically enhance the protective immune responses of PCV2 adenovirus vaccine, which would be used as a potent vaccine for the prevention and control of PCVAD.

Agreement of skin test with IL-4 production and CD40L expression by T cells upon immunotherapy of subjects with systemic reactions to Hymenoptera stings.

Venom immunotherapy is the only curative intervention for subjects with Hymenoptera venom allergy who suffering systemic reactions upon bee or wasp stings. Venom immunotherapy can restore normal immunity against venom allergens, as well as providing to allergic subjects a lifetime tolerance against venoms. Nevertheless, it is necessary using safety assays to monitoring the development of tolerance in the VIT protocols to avoid fatal anaphylactic reactions. The purpose of this study was to assess the modifications in several markers of tolerance induction in subjects with Hymenoptera venom allergy undergoing immunotherapy. The studies were performed at baseline time and after six month of VIT. Intradermal skin tests, basophil activation tests, specific IgE levels; and the T-cell markers (IL-4 and IFN-γ producing cells; and expression of the surface activation markers CD40L and CTLA-4) were assayed. At six month of immunotherapy all parameters studied had significant alterations. All decreased, except the IFN-γ producing cells. In addition, modifications in intradermal skin test showed a significant correlation with both, CD40L expression on CD4 T lymphocytes (p=0.043) and IL-4 producing T lymphocytes (p=0.012). Neither basophil activation test nor serum levels of sIgE demonstrated any correlation with the immunological parameters studied nor among them. These results suggest that both IL-4 production and CD40L expression could be two good indicators of the beneficial effects of venom immunotherapy which translate into skin tests.

Expression of CD154 (CD40L) on stimulated T lymphocytes in patients with idopathic thrombocytopenic purpura.

OBJECTIVES: The role of CD40-CD154 (CD40L) interaction in T cell-dependent humoral immune response is strongly established. Increased expression of CD154 on stimulated T cells is observed in rheumatic diseases and is associated with disease activity. We investigated the expression of CD154 on T cells from idiopathic thrombocytopenic purpura (ITP) patients and assessed the significance of CD154 expression in disease status. METHODS: We enrolled 59 ITP patients, 23 healthy controls, and 19 patients with non-immune thrombocytopenia. Isolated mononuclear cells were stimulated in RPMI medium containing phorbol myristate acetate (PMA) (5 ng/mL) and ionomycin (500 ng/mL) for 2 h at 37°C. The expression of CD154 on CD4(+)T cells was evaluated using flow cytometry and serum soluble CD40L levels were measured. RESULTS: In ITP patients, the percentage of CD4(+) CD154(+) cells and mean fluorescence intensity (MFI) of CD154 on activated CD4(+)T cells was not different from that in the healthy controls and non-immune thrombocytopenia patients. Additionally, the percentage and expression level of CD154 was not different between ITP patients with low platelet counts (<50 000/µL) and those with 50 000/µL or more. Soluble CD40L levels were significantly lower in ITP patients with low platelet counts than in healthy controls, but were not correlated with CD154 expression. CONCLUSION: Increased CD154 expression on CD4(+)T cells was not observed in ITP patients and was not related with low platelet counts. Overexpression of CD154 on CD4(+)T cells is unlikely to be central to the pathogenesis of ITP, and other immune dysfunctions should be targeted for therapy purposes.

T Cell Responses against Mycobacterial Lipids and Proteins Are Poorly Correlated in South African Adolescents.

Human T cells are activated by both peptide and nonpeptide Ags produced by Mycobacterium tuberculosis. T cells recognize cell wall lipids bound to CD1 molecules, but effector functions of CD1-reactive T cells have not been systematically assessed in M. tuberculosis-infected humans. It is also not known how these features correlate with T cell responses to secreted protein Ags. We developed a flow cytometric assay to profile CD1-restricted T cells ex vivo and assessed T cell responses to five cell wall lipid Ags in a cross-sectional study of 19 M. tuberculosis-infected and 22 M. tuberculosis-uninfected South African adolescents. We analyzed six T cell functions using a recently developed computational approach for flow cytometry data in high dimensions. We compared these data with T cell responses to five protein Ags in the same cohort. We show that CD1b-restricted T cells producing antimycobacterial cytokines IFN-γ and TNF-α are detectable ex vivo in CD4(+), CD8(+), and CD4(-)CD8(-) T cell subsets. Glucose monomycolate was immunodominant among lipid Ags tested, and polyfunctional CD4 T cells specific for this lipid simultaneously expressed CD40L, IFN-γ, IL-2, and TNF-α. Lipid-reactive CD4(+) T cells were detectable at frequencies of 0.001-0.01%, and this did not differ by M. tuberculosis infection status. Finally, CD4 T cell responses to lipids were poorly correlated with CD4 T cell responses to proteins (Spearman rank correlation -0.01; p = 0.95). These results highlight the functional diversity of CD1-restricted T cells circulating in peripheral blood as well as the complementary nature of T cell responses to mycobacterial lipids and proteins. Our approach enables further population-based studies of lipid-specific T cell responses during natural infection and vaccination.

Hyperactivation and in situ recruitment of inflammatory Vδ2 T cells contributes to disease pathogenesis in systemic lupus erythematosus.

In this study, we measured the proportion of peripheral Vδ2 T cells as well as the status and chemokine receptor expression profiles in SLE patients and healthy control (HC). In addition, Vδ2 T cell infiltration in the kidneys of patients with lupus nephritis was examined. The results showed that the percentage of peripheral Vδ2 T cells in new-onset SLE was decreased, and negatively correlated with the SLE Disease Activity Index score and the severity of proteinuria. These cells had a decreased apoptosis but an increased proliferation, and they showed increased accumulation in SLE kidneys. Moreover, IL-21 production and CD40L, CCR4, CCR7, CCR8, CXCR1 and CX3CR1 expression in Vδ2 T cells from SLE patients was significantly higher than from HC (p < 0.05), and these factors were downregulated in association with the repopulation of peripheral Vδ2 T cells in patients who were in remission (p < 0.05). In addition, anti-TCR Vδ2 antibodies activation significantly upregulated these chemokine receptors on Vδ2 T cells from HC, and this effect was blocked by inhibitors of PLC-γ1, MAPK/Erk, and PI3K signaling pathways. Our findings demonstrate that the distribution and function status of Vδ2 T cells from SLE patients are abnormal, and these aberrations may contribute to disease pathogenesis.

Inhibition of FOXP3/NFAT Interaction Enhances T Cell Function after TCR Stimulation.

Regulatory T cell (Treg) activity is modulated by a cooperative complex between the transcription factor NFAT and FOXP3, a lineage specification factor for Tregs. FOXP3/NFAT interaction is required to repress expression of IL-2, upregulate expression of the Treg markers CTLA4 and CD25, and confer suppressor function to Tregs. However, FOXP3 is expressed transiently in conventional CD4(+) T cells upon TCR stimulation and may lead to T cell hyporesponsiveness. We found that a short synthetic peptide able to inhibit FOXP3/NFAT interaction impaired suppressor activity of conventional Tregs in vitro. Specific inhibition of FOXP3/NFAT interaction with this inhibitory peptide revealed that FOXP3 downregulates NFAT-driven promoter activity of CD40L and IL-17. Inhibition of FOXP3/NFAT interaction upregulated CD40L expression on effector T cells and enhanced T cell proliferation and IL-2, IFN-γ, IL-6, or IL-17 production in response to TCR stimulation. The inhibitory peptide impaired effector T cell conversion into induced Tregs in the presence of TGF-ß. Moreover, in vivo peptide administration showed antitumor efficacy in mice bearing Hepa129 or TC1 tumor cells when combined with sorafenib or with an antitumor vaccine, respectively. Our results suggest that inhibition of NFAT/FOXP3 interaction might improve antitumor immunotherapies.

Vaccination Produces CD4 T Cells with a Novel CD154-CD40-Dependent Cytolytic Mechanism.

The discovery of new vaccines against infectious diseases and cancer requires the development of novel adjuvants with well-defined activities. The TLR4 agonist adjuvant GLA-SE elicits robust Th1 responses to a variety of vaccine Ags and is in clinical development for both infectious diseases and cancer. We demonstrate that immunization with a recombinant protein Ag and GLA-SE also induces granzyme A expression in CD4 T cells and produces cytolytic cells that can be detected in vivo. Surprisingly, these in vivo CTLs were CD4 T cells, not CD8 T cells, and this cytolytic activity was not dependent on granzyme A/B or perforin. Unlike previously reported CD4 CTLs, the transcription factors Tbet and Eomes were not necessary for their development. CTL activity was also independent of the Fas ligand-Fas, TRAIL-DR5, and canonical death pathways, indicating a novel mechanism of CTL activity. Rather, the in vivo CD4 CTL activity induced by vaccination required T cell expression of CD154 (CD40L) and target cell expression of CD40. Thus, vaccination with a TLR4 agonist adjuvant induces CD4 CTLs, which kill through a previously unknown CD154-dependent mechanism.

CD40/CD40L expression correlates with the survival of patients with glioblastomas and an augmentation in CD40 signaling enhances the efficacy of vaccinations against glioma models.

BACKGROUND: The prognosis of glioblastoma (GBM) remains poor; therefore, effective therapeutic strategies need to be developed. CD40 is a costimulatory molecule whose agonistic antibody has been shown to activate antitumor effects. Recently, CD40 has been extensively targeted for immunotherapeutic purposes. METHODS: Expressions of CD40/CD40L mRNAs were examined in 86 cases of World Health Organization grade IV GBM and 36 cases of grade III gliomas and correlated with outcomes. CD40 signaling was employed to augment the efficacy of immunotherapy against gliomas. The efficacy of FGK45, an agonistic antibody for CD40, was examined by adding it to a tumor lysate-based subcutaneous vaccination against a GL261 glioma model and an NSCL61 glioma-initiating cell-like cell tumor model. RESULTS: We demonstrated for the first time using quantitative PCR that grade III gliomas express higher levels of CD40/CD40L than does grade IV GBM. The higher expression of CD40/CD40L was associated with good prognoses in patients with GBM. Addition of FGK45 to the subcutaneous tumor cell lysate-based vaccination significantly prolonged survival in both tumor models. However, the efficacy was modest in NSCL61-model mice. Therefore, we established combination immunotherapeutic strategies using FGK45 and OX86, an agonistic antibody for OX40. Combination immunotherapy significantly prolonged survival with synergistic effects. Apoptosis increased and proliferation decreased in tumors treated with combination immunotherapy. CONCLUSIONS: The high expression of CD40/CD40L can be used as a biomarker for better prognoses in patients with gliomas. Immunotherapy using FGK45 significantly prolonged survival and represents a potential therapeutic strategy for gliomas including glioma-initiating cells.

The expression of sphingosine-1 phosphate receptor-1 in chronic lymphocytic leukemia cells is impaired by tumor microenvironmental signals and enhanced by piceatannol and R406.

Chronic lymphocytic leukemia (CLL) is characterized by the progressive accumulation of clonal B lymphocytes. Proliferation occurs in lymphoid tissues upon interaction of leukemic cells with a supportive microenvironment. Therefore, the mobilization of tissue-resident CLL cells into the circulation is a useful therapeutic strategy to minimize the reservoir of tumor cells within survival niches. Because the exit of normal lymphocytes from lymphoid tissues depends on the presence of sphingosine-1 phosphate (S1P) and the regulated expression of S1P receptor-1 (S1PR1), we investigated whether the expression and function of S1PR1 can be modulated by key microenvironment signals. We found that activation of CLL cells with CXCL12, fibroblast CD40L(+), BCR cross-linking, or autologous nurse-like cells reduces their S1PR1 expression and the migratory response toward S1P. Moreover, we found that S1PR1 expression was reduced in the proliferative/activated subset of leukemic cells compared with the quiescent subset from the same patient. Similarly, bone marrow-resident CLL cells expressing high levels of the activation marker CD38 showed a lower expression of S1PR1 compared with CD38(low) counterparts. Finally, given that treatment with BCR-associated kinase inhibitors induces a transient redistribution of leukemic cells from lymphoid tissues to circulation, we studied the effect of the Syk inhibitors piceatannol and R406 on S1PR1 expression and function. We found that they enhance S1PR1 expression in CLL cells and their migratory response toward S1P. Based on our results, we suggest that the regulated expression of S1PR1 might modulate the egress of the leukemic clone from lymphoid tissues.