Exogenous IL-33 Administration Research Articles

Anti-Vascular Endothelial Growth Factor Therapy Abolishes Glioma-Associated Endothelial Cell-Induced Tumor Invasion.

Tumor-remodeled endothelial cells not only facilitate the formation of tumor angiogenesis but also promote tumorigenesis. In this study, we aimed to explore the interaction between glioma-associated endothelial cells (GAEs) and glioma cells. We found that different subtypes of glioma owned distinct GAE abundance. Glioma patients with high GAE abundance exhibited poor prognosis. Both the results of the bioinformatics analysis and the in vitro co-culture system assay revealed that GAE promoted glioma cell invasion. Besides, anti-vascular endothelial growth factor (VEGF) therapy partially abolished the effects of GAE on gliomas. Moreover, anti-VEGF therapy upregulated IL-2 expression in GAE, and exogenous IL-2 administration inhibits GAE-induced glioma cell invasion. Collectively, our present study provides a novel outstanding of the interaction between GAE and glioma cells.

Recombinant Expression of Human IL-33 Protein and Its Effect on Skin Wound Healing in Diabetic Mice.

Chronic refractory wounds are one of the complications of diabetes mellitus that require effective therapy. The dermal-wound-healing property of IL-33 in diabetics is little understood. Therefore, this study aimed to express recombinant humanized mature IL-33 (rhmatIL-33) in Escherichia coli BL21 (DE3) and demonstrate its efficacy on dermal wounds in streptozotocin (STZ)-induced diabetic and nondiabetic mice by the dorsal incisional skin wound model. Results revealed that the rhmatIL-33 accelerated the scratch-healing of keratinocytes and fibroblasts at the cellular level. The wounds of diabetic mice (DM) showed more severe ulceration and inflammation than wild-type mice (WT), and the exogenous administration of rhmatIL-33 increased wound healing in both diabetic and wild-type mice. Compared with the up-regulation of endogenous IL-33 mRNA after injury in WT mice, the IL-33 mRNA decreased after injury in DM mice. Exogenous IL-33 administration increased the endogenous IL-33 mRNA in the DM group but decreased the IL-33 mRNA expression level of the WT group, indicating that IL-33 plays a balancing role in wound healing. IL-33 administration also elevated ILC2 cells in the wounds of diabetic and non-diabetic mice and improve the transcript levels of YM1, a marker of M2 macrophages. In conclusion, Hyperglycemia in diabetic mice inhibited the expression of IL-33 in the dermal wound. Exogenous addition of recombinant IL-33 promoted wound healing in diabetic mice by effectively increasing the level of IL-33 in wound tissue, increasing ILC2 cells, and accelerating the transformation of macrophage M1 to M2 phenotype.

IL-33 ameliorates liver injury and inflammation in Poly I:C and Concanavalin-A induced acute hepatitis

The IL-33/ST2 axis is known to be involved in liver pathologies and IL-33 is over-expressed in mouse hepatitis models. We aimed to investigate the proposed protective effect of IL-33 in murine fulminant hepatitis induced by a Toll like receptor 3 (TLR3) viral mimetic, Poly I:C or by Concanavalin-A (ConA). The Balb/C mice were administered intravenously with ConA (15 mg/kg) or Poly I:C (30 μg/mouse) to induce acute hepatitis along with vehicle control. The recombinant mouse IL-33 (rIL-33) was injected (0.2 μg/mouse) to mice 2 h prior to ConA or Poly I:C injection to check its hepato-protective effects. The gross lesions, level of serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), histopathology (H&E staining) and levels of IFNγ and TNFα were measured by ELISA. The gross pathological liver injury induced by Poly I:C or ConA was reduced by rIL-33 administration in mice. The levels of AST and ALT were significantly (P ≤ 0.05) higher in mice challenged with Poly I:C or ConA in comparison to control mice. The rIL-33 pre-treated mice in both Poly I:C and ConA challenge groups showed significantly (P ≤ 0.05) lower levels of AST and ALT, and decreased liver injury (parenchymal and per-vascular necrotic areas) in histological liver sections. The soluble levels of TNFα and IFNγ were significantly (P ≤ 0.05) raised in Poly I:C or ConA challenged mice than control mice. The levels of TNFα and IFNγ were significantly reduced (P ≤ 0.05) in rIL-33 pre-treated mice. In conclusion, the exogenous IL-33 administration mitigated liver injury and inflammation (decreased levels of IFNγ and TNFα) in Poly I:C and ConA-induced acute hepatitis in mice.

IκBα Nuclear Export Enables 4-1BB-Induced cRel Activation and IL-2 Production to Promote CD8 T Cell Immunity.

Optimal CD8 T cell immunity is orchestrated by signaling events initiated by TCR recognition of peptide Ag in concert with signals from molecules such as CD28 and 4-1BB. The molecular mechanisms underlying the temporal and spatial signaling dynamics in CD8 T cells remain incompletely understood. In this study, we show that stimulation of naive CD8 T cells with agonistic CD3 and CD28 Abs, mimicking TCR and costimulatory signals, coordinately induces 4-1BB and cRel to enable elevated cytosolic cRel:IκBα complex formation and subsequent 4-1BB-induced IκBα degradation, sustained cRel activation, heightened IL-2 production and T cell expansion. NfkbiaNES/NES CD8 T cells harboring a mutated IκBα nuclear export sequence abnormally accumulate inactive cRel:IκBα complexes in the nucleus following stimulation with agonistic anti-CD3 and anti-CD28 Abs, rendering them resistant to 4-1BB induced signaling and a disrupted chain of events necessary for efficient T cell expansion. Consequently, CD8 T cells in NfkbiaNES/NES mice poorly expand during viral infection, and this can be overcome by exogenous IL-2 administration. Consistent with cell-based data, adoptive transfer experiments demonstrated that the antiviral CD8 T cell defect in NfkbiaNES/NES mice was cell intrinsic. Thus, these results reveal that IκBα, via its unique nuclear export function, enables, rather than inhibits 4-1BB-induced cRel activation and IL-2 production to facilitate optimal CD8 T cell immunity.

Abstract B84: Decipher the role of IL-33 as an activator of NK cells’ antitumor activity

Abstract Interleukin (IL)-33 is an alarmin belonging to the IL-1 family. Through its receptor ST2, IL-33 promotes both type 1 and type 2 immune responses, depending on the type of responding cells and the microenvironment in damaged tissues. Consistently, paradoxical roles of IL-33 have been reported in the context of cancer. These observations prompted us to investigate i) the expression of IL-33/ST2 in breast tumors and ii) the role of IL-33/ST2 in NK cell activation in physiologic and tumor contexts. Using immunohistochemistry (IHC) and transcriptomic analyses, we observed that IL-33 is expressed at higher levels in ductal carcinoma in situ (DCIS) compared to invasive breast cancer (IBC), especially in the stroma and in luminal BC subtype. In situ, IL-33 was mainly detected in endothelial cells and at lower extent in scattered cells within the stroma of breast tumors. Furthermore, we unraveled a new pathway for NK cells’ activation where IL-12 upregulates ST2 on human NK cells, which in turn become responsive to IL-33 by secreting high levels of IFN-γ and increasing their cytotoxic activity. This effect was specific to a subset of human CD56dim NK cells (20%) and was dependent on STAT-4 phosphorylation. In mice, we also observed a strong activation of spleen NK cell by IL-33 in combination with IL-12 in vitro. Finally, using IL-33ko mice, preliminary results show a contribution of endogenous IL-33 in the prevention of experimental lung metastasis development following B16 iv injection in therapeutic settings using exogenous administration of IL-12. Following up on these results, our aim is now to i) better characterize IL-33-responsive NK cells subpopulation in blood, ii) understand why human CD56bright NK cells do not upregulate ST2 despite response to IL-12 by phosphorylating STAT-4, iii) characterize NK cells’ response to IL-33 in human breast tumors, iv) evaluate ST2 expression by immune cells infiltrating breast and ovarian tumors, and v) confirm the activating role of IL-33 on NK cells’ biology in tumor models in vivo. All together, our observations are in favor of an NK-mediated antitumor role of IL-33 that we are currently pursuing as a potential novel therapeutic strategy in cancer. Citation Format: Anais Eberhardt, Elena Blanc, Emilie Lardenois, Sarah Renaudineau, Jennifer Herbulot, Benoit Dumont, Isabelle Durand, Emilie Charrier, Pauline Schmitt, Jean-Philippe Girard, Antoine Marçais, Thierry Walzer, Christophe Caux, Nathalie Bendriss-Vermare. Decipher the role of IL-33 as an activator of NK cells’ antitumor activity [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B84.

Focused Ultrasound Improves NK-92MI Cells Infiltration Into Tumors.

The efficiency of natural killer (NK) cells, adoptively transferred, for treatment against solid tumors is hindered by their difficulty to enter tumors from the blood circulation as well as their inability to prolong viability in the absence of IL-2. Among different sources of NK cells, we used genetically modified NK-92MI cells, a suitable candidate which can release IL-2 to maintain their viability and overcome undesirable side effects caused by systemic administration of exogenous IL-2. In this study, we evaluated whether the combination of focused ultrasound (FUS) and microbubbles can improve adoptively NK-92MI cell infiltration into ovarian tumors through biodistribution, immunofluorescence, and flow cytometry. The treatment effects of using this strategy twice a week were explored. The potential molecular mechanism of FUS assisting NK cell therapy was also initially explored through evaluating the expression of ICAM1 and CX3CL1 by qRT-PCR. Our results indicated that FUS and microbubbles can improve NK-92MI cells’ infiltration into tumors, and the combination of FUS and NK-92MI cells had a better treatment effect compared to the PBS group, but not compared to the NK-92MI group. The qRT-PCR results also showed that CX3CL1 may be involved in the process of FUS-assisted NK cell infiltration. These results indicate that further optimization of the FUS-assisted strategy is still needed to achieve therapeutic benefit.

IL-33 promotes recovery from acute colitis by inducing miR-320 to stimulate epithelial restitution and repair

Defective and/or delayed wound healing has been implicated in the pathogenesis of several chronic inflammatory disorders, including inflammatory bowel disease (IBD). The resolution of inflammation is particularly important in mucosal organs, such as the gut, where restoration of epithelial barrier function is critical to reestablish homeostasis with the interfacing microenvironment. Although IL-33 and its receptor ST2/ILRL1 are known to be increased and associated with IBD, studies using animal models of colitis to address the mechanism have yielded ambiguous results, suggesting both pathogenic and protective functions. Unlike those previously published studies, we focused on the functional role of IL-33/ST2 during an extended (2-wk) recovery period after initial challenge in dextran sodium sulfate (DSS)-induced colitic mice. Our results show that during acute, resolving colitis the normal function of endogenous IL-33 is protection, and the lack of either IL-33 or ST2 impedes the overall recovery process, while exogenous IL-33 administration during recovery dramatically accelerates epithelial restitution and repair, with concomitant improvement of colonic inflammation. Mechanistically, we show that IL-33 stimulates the expression of a network of microRNAs (miRs) in the Caco2 colonic intestinal epithelial cell (IEC) line, especially miR-320, which is increased by >16-fold in IECs isolated from IL-33-treated vs. vehicle-treated DSS colitic mice. Finally, IL-33-dependent in vitro proliferation and wound closure of Caco-2 IECs is significantly abrogated after specific inhibition of miR-320A. Together, our data indicate that during acute, resolving colitis, IL-33/ST2 plays a crucial role in gut mucosal healing by inducing epithelial-derived miR-320 that promotes epithelial repair/restitution and the resolution of inflammation.

Transcription factor c-Rel is indispensable for generation of thymic but not of peripheral Foxp3+ regulatory T cells.

The transcription factor c-Rel has been shown to be crucial for development of regulatory T cells (Tregs). Recent studies have reported that the expression of transcription factor Helios in Foxp3+ Tregs correlates with thymic origin of these cells (tTregs). Notably, we found that only the Helios+Foxp3+ Treg cell population was substantially reduced in c-Rel deficient mice. In contrast to a defective tTreg development, we observed an expansion of mucosal Tregs during the induction of acute colitis in rel−/− mice. Furthermore, we found a preferential accumulation of Helios−Foxp3+ Tregs in aged c-Rel deficient mice. This unexpected finding, together with the observation that naïve CD4+ T cells convert into Tregs in vitro in the absence of c-Rel and presence of IL-2, provide an evidence that extra-thymic generation of induced and peripheral Tregs (iTregs and pTregs) is independent of c-Rel. Moreover, the treatment with IL-2/anti-IL-2 mAb (JES6-1) resulted in a widespread increase of Helios+Foxp3+ Tregs in both wild-type (WT) and rel−/− mice. These data suggest that exogenous IL-2 administration compensates for defective IL-2 production and reduced tTreg numbers in c-Rel deficient mice. Our findings reveal that c-Rel is essential for the generation of tTregs but not for that of pTregs and iTregs.

Adenovirus Serotype 5 Vaccination Results in Suboptimal CD4 T Helper 1 Responses in Mice.

Adenovirus serotype 5 (Ad5) is one of the most widely used viral vectors and is known to generate potent T cell responses. While many previous studies have characterized Ad5-induced CD8 T cell responses, there is a relative lack of detailed studies that have analyzed CD4 T cells elicited by Ad5 vaccination. Here, we immunized mice with Ad5 vectors encoding lymphocytic choriomeningitis virus (LCMV) glycoprotein (GP) and examined GP-specific CD4 T cell responses elicited by Ad5 vectors and compared them to those induced by an acute LCMV infection. In contrast to LCMV infection, where balanced CD4 T helper 1 (Th1) and T follicular helper (Tfh) responses were induced, Ad5 immunization resulted in a significantly reduced frequency of Th1 cells. CD4 T cells elicited by Ad5 vectors expressed decreased levels of Th1 markers, such as Tim3, SLAM, T-bet, and Ly6C, had smaller amounts of cytotoxic molecules like granzyme B, and produced less interferon gamma than CD4 T cells induced by LCMV infection. This defective CD4 Th1 response appeared to be intrinsic for Ad5 vectors and not a reflection of comparing a nonreplicating vector to a live viral infection, since immunization with a DNA vector expressing LCMV-GP generated efficient CD4 Th1 responses. Analysis at early time points (day 3 or 4) after immunization with Ad5 vectors revealed a defect in the expression of CD25 (interleukin-2 [IL-2] receptor alpha chain) on Ad5-elicited CD4 T cells, and administration of exogenous IL-2 following Ad5 immunization partially restored CD4 Th1 responses. These results suggest that impairment of Th1 commitment after Ad5 immunization could be due to reduced IL-2-mediated signaling.IMPORTANCE During viral infection, generating balanced responses of Th1 and Tfh cells is important to induce effective cell-mediated responses and provide optimal help for antibody responses. In this study, to investigate vaccine-induced CD4 T cell responses, we characterized CD4 T cells after immunization with Ad5 vectors expressing LCMV-GP in mice. Ad5 vectors led to altered effector differentiation of LCMV GP-specific CD4 T cells compared to that during LCMV infection. CD4 T cells following Ad5 immunization exhibited impaired Th1 lineage commitment, generating significantly decreased Th1 responses than those induced by LCMV infection. Our results suggest that suboptimal IL-2 signaling possibly plays a role in reduced Th1 development following Ad5 immunization.

IL-33 Precedes IL-5 in Regulating Eosinophil Commitment and Is Required for Eosinophil Homeostasis.

Eosinophils are important in the pathogenesis of many diseases, including asthma, eosinophilic esophagitis, and eczema. Whereas IL-5 is crucial for supporting mature eosinophils (EoMs), the signals that support earlier eosinophil lineage events are less defined. The IL-33R, ST2, is expressed on several inflammatory cells, including eosinophils, and is best characterized for its role during the initiation of allergic responses in peripheral tissues. Recently, ST2 expression was described on hematopoietic progenitor subsets, where its function remains controversial. Our findings demonstrate that IL-33 is required for basal eosinophil homeostasis, because both IL-33- and ST2-deficient mice exhibited diminished peripheral blood eosinophil numbers at baseline. Exogenous IL-33 administration increased EoMs in both the bone marrow and the periphery in wild-type and IL-33-deficient, but not ST2-deficient, mice. Systemic IL-5 was also increased under this treatment, and blocking IL-5 with a neutralizing Ab ablated the IL-33-induced EoM expansion. The homeostatic hypereosinophilia seen in IL-5-transgenic mice was significantly lower with ST2 deficiency despite similar elevations in systemic IL-5. Finally, in vitro treatment of bone marrow cells with IL-33, but not IL-5, led to specific early expansion of IL-5Rα-expressing precursor cells. In summary, our findings establish a basal defect in eosinophilopoiesis in IL-33- and ST2-deficient mice and a mechanism whereby IL-33 supports EoMs by driving both systemic IL-5 production and the expansion of IL-5Rα-expressing precursor cells.

IL-33 Drives Eosinophil Infiltration and Pathogenic Type 2 Helper T-Cell Immune Responses Leading to Chronic Experimental Ileitis

Although a clear association has been established between IL-33 and inflammatory bowel disease, mechanistic studies to date, primarily using acute murine models of colitis, have yielded contradicting results, demonstrating both pathogenic and protective roles. We used a well-characterized, spontaneous model of inflammatory bowel disease [ie, SAMP1/YitFc (SAMP) mice] to investigate the role of IL-33 during chronic intestinal inflammation. Our results showed marked eosinophil infiltration into the gut mucosa with increased levels of eotaxins and type 2 helper T-cell (Th2) cytokines as disease progressed and became more severe, which could be reversed upon either eosinophil depletion or blockade of IL-33 signaling. Exogenous IL-33 administration recapitulated these effects in ilea of uninflamed (parental) control AKR/J mice. Human data supported these findings, showing colocalization and up-regulation of IL-33 and eosinophils in the colonic mucosa of inflammatory bowel disease patients versus noninflamed controls. Finally, colonization of commensal flora by fecal material transplantation into germ-free SAMP and the presence of the gut microbiome induced IL-33, subsequent eosinophil infiltration, and mounting of Th2 immune responses, leading to exacerbation of chronic intestinal inflammation characteristic of SAMP mice. These data demonstrate a pathogenic role for IL-33-mediated eosinophilia and activation of Th2 immunity in chronic intestinal inflammation that is dependent on the gut microbiome. Targeting IL-33 may represent a novel therapeutic approach to treat patients with inflammatory bowel disease.

Abstract 1340: Antitumor effector B cells directly kill tumor cells involving the CXCL12/CXCR4 pathway and their therapeutic efficacy is enhanced by IL-2

Abstract In cancer immunotherapy, effector B cells can inhibit spontaneous tumor metastases as well as local tumor growth in animal models. We have previously reported that antitumor effector B cells generated from tumor-draining lymph node (TDLN) could directly kill tumor cells via the Fas/FasL pathway and are regulated by IL-10. Trafficking of TDLN B cells in vivo suggested that they were recruited to the tumor and lung as well as secondary lymphoid organs. The number of TDLN B cells increased following transfer. As a result, adoptive transfer of TDLN B cells conferred tumor regression in a spontaneous pulmonary metastasis mouse model of breast cancer, 4T1. Removal of IL-10, either by using IL-10-/- TDLN B cells or by systemic neutralization of IL-10 using anti-IL-10 antibody, significantly augmented the therapeutic efficacy of adoptively transferred TDLN B cells. In recent studies, we found that tumor cell death mediated by effector B cells involves the CXCR4/CXCL12 as well as Fas/FasL interactions. Blockade of CXCR4 and FasL using a CXCR4-specific inhibitor, AMD3100, and anti-FasL antibody, respectively, reduced the killing of 4T1 tumor cells by 4T1 TDLN B cells. Of note, blockade of CXCR4 and FasL concurrently inhibited B cell-mediated direct killing of tumor cells in an additive fashion, suggesting that both CXCL12/CXCR4 and Fas/FasL pathways are involved in the direct killing of 4T1 tumor cells by 4T1 TDLN B cells. In other experiments, we found that TDLN B cells express IL-2 receptors (IL-2R) and the ability of the TDLN B cells to inhibit metastasis was significantly enhanced with exogenous IL-2 administration. Besides our previously reported Fas/FasL pathway and IL-10-mediated suppression, our recently identified CXCL12/CXCR4 pathway and IL-2-mediated augmentation of the biological function of effector B cells suggests these B cells may represent an important component of the host immune response against tumor. Citation Format: Yang Xia, Huimin Tao, Yangyang Hu, Li Zhou, Yangyi Bao, Steven K Lundy, Shiang Huang, Qiao Li, Alfred E Chang. Antitumor effector B cells directly kill tumor cells involving the CXCL12/CXCR4 pathway and their therapeutic efficacy is enhanced by IL-2. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1340. doi:10.1158/1538-7445.AM2015-1340

Repeated Injections of IL-2 Break Renal Allograft Tolerance Induced via Mixed Hematopoietic Chimerism in Monkeys.

Tolerance of allografts achieved in mice via stable mixed hematopoietic chimerism relies essentially on continuous elimination of developing alloreactive T cells in the thymus (central deletion). Conversely, while only transient mixed chimerism is observed in nonhuman primates and patients, it is sufficient to ensure tolerance of kidney allografts. In this setting, it is likely that tolerance depends on peripheral regulatory mechanisms rather than thymic deletion. This implies that, in primates, upsetting the balance between inflammatory and regulatory alloimmunity could abolish tolerance and trigger the rejection of previously accepted renal allografts. In this study, six monkeys that were treated with a mixed chimerism protocol and had accepted a kidney allograft for periods of 1-10 years after withdrawal of immunosuppression received subcutaneous injections of IL-2 cytokine (0.6-3 × 10(6) IU/m(2) ). This resulted in rapid rejection of previously tolerated renal transplants and was associated with an expansion and reactivation of alloreactive pro-inflammatory memory T cells in the host's lymphoid organs and in the graft. This phenomenon was prevented by anti-CD8 antibody treatment. Finally, this process was reversible in that cessation of IL-2 administration aborted the rejection process and restored normal kidney graft function.

The IL-33/ST2 axis augments effector T-cell responses during acute GVHD

AbstractInterleukin (IL)-33 binding to the receptor suppression of tumorigenicity 2 (ST2) produces pro-inflammatory and anti-inflammatory effects. Increased levels of soluble ST2 (sST2) are a biomarker for steroid-refractory graft-versus-host disease (GVHD) and mortality. However, whether sST2 has a role as an immune modulator or only as a biomarker during GVHD was unclear. We show increased IL-33 production by nonhematopoietic cells in the gastrointestinal (GI) tract in mice post-conditioning and patients during GVHD. Exogenous IL-33 administration during the peak inflammatory response worsened GVHD. Conversely, GVHD lethality and tumor necrosis factor-α production was significantly reduced in il33−/− recipients. ST2 was upregulated on murine and human alloreactive T cells and sST2 increased as experimental GVHD progressed. Concordantly, st2−/− vs wild-type (WT) donor T cells had a marked reduction in GVHD lethality and GI histopathology. Alloantigen-induced IL-18 receptor upregulation was lower in st2−/− T cells, and linked to reduced interferon-γ production by st2−/− vs WT T cells during GVHD. Blockade of IL-33/ST2 interactions during allogeneic-hematopoietic cell transplantation by exogenous ST2-Fc infusions had a marked reduction in GVHD lethality, indicating a role of ST2 as a decoy receptor modulating GVHD. Together, these studies point to the IL-33/ST2 axis as a novel and potent target for GVHD therapy.

Interleukin-33 promotes Th2 immune responses in infected mice with Schistosoma japonicum

IL-33, a new member of the IL-1 cytokine family, is associated with many infectious diseases. IL-33 not only is crucial for induction of Th2 polarized responses, but also is involved in induction of inflammation as a proinflammatory cytokine. Whether IL-33 leads to beneficial or worsening outcomes depends on the immune mechanism underlying the pathogensis of each disease condition. This study was to elucidate the role of IL-33 in schistosomiasis japonica in a mouse model. Our results demonstrated that serum levels of IL-33 from infected mice with Schistosoma japonicum began to rise at 1 week postinfection (pi) and reached a peak in 7 weeks pi, and then remained a plateau for 2 weeks, after which its level gradually decreased until 12 weeks pi. Compared with the infection control, exogenous IL-33 administration could increase a Th2 polarized immune response (evidenced by higher levels of IL-5, IL-10, and IL-13, along with lower level of IFN-γ) at 6 weeks pi. Meanwhile, this Th2 polarization was associated with higher infection intensity and liver immunopathology in infected mice, whereas injection of anti-IL-33 mAb into infected mice induced adverse effects on these above immune parameters and immunopathology. These data suggest that IL-33 might act as an inducer of Th2 polarization and plays a crucial role in immunopathology in murine schistosomiasis japonica.

The anergy induction of M3 muscarinic acetylcholine receptor-reactive CD4+ T cells suppresses experimental sialadenitis-like Sjögren's syndrome.

Autoreactive CD4+ T cells are involved in the pathogenesis of Sjögren's syndrome (SS). The aim of the present study was to clarify the dominant T cell epitopes of M3 muscarinic acetylcholine receptor (M3R) and to establish a new antigen-specific therapy for SS using an experimental mouse model. Production of cytokines from M3R-reactive CD4+ T cells, after culture with various M3R peptides, was analyzed by enzyme-linked immunosorbent assay. Adoptive cell transfer was performed using splenocytes from M3R(-/-) mice that were immunized with M3R peptides or phosphate buffered saline plus H37Ra as a control. Rag1(-/-) mice were inoculated with the splenocytes and examined for the development of sialadenitis. Altered peptide ligands (APLs) of the T cell epitopes, with substitutions in amino acid residues at T cell receptor contact sites, were synthesized, and the ability of the APLs to suppress sialadenitis was evaluated. The mechanisms underlying such effects were assessed. CD4+ M3R-reactive T cells produced interleukin-17 (IL-17) and interferon-γ (IFNγ) in response to the N-terminal 1 (N1) and 1st extracellular loop peptides of M3R, and Rag1(-/-) mice that received N1- and/or 1st peptide-immunized splenocytes developed sialadenitis. Among the designed APLs, N1-APL7 (N→S at amino acid 15) significantly suppressed IFNγ production in vitro, and also suppressed sialadenitis in vivo. Levels of early growth response 2 in CD4+ T cells from the cervical lymph nodes of N1-APL7-treated mice were significantly higher than those of control mice, and cell proliferation was reversed by administration of exogenous IL-2. Levels of the anergy-related molecules itchy homolog E3 ubiquitin-protein ligase, Casitas B-lineage lymphoma b, gene related to anergy in lymphocytes, and Deltex-1 were significantly higher in CD4+ T cells cultured with N1-APL7. The major T cell epitopes were from the N1 and 1st peptide regions. Moreover, N1-APL7, selected as the antagonistic APL in vitro, also suppressed sialadenitis through the induction of anergy. This is a potentially useful strategy for regulating pathogenic T cell infiltration in SS.

Common gamma chain (γc) cytokines differentially potentiate TNFR family signaling in antigen-activated CD8(+) T cells.

BackgroundSeveral members of the common gamma chain (gc) cytokine family are already approved (IL-2) or actively being developed as vaccine adjuvants and cancer immunotherapies. Studies have indicated that co-administration of gc cytokines may enhance the efficacy of immunotherapies that function via direct activation of co-stimulatory T cell receptors. To define the specific influence of gc cytokines on the co-stimulatory capacity of CD8+ T cells and identify combinations with synergistic potential, we investigated the direct impact of gc cytokines on the differentiation and transcriptional profile of recently antigen-primed CD8+ T cells.MethodsNaïve CD8+ T cells were activated with peptide-pulsed APCs. After 48 hours, CD8+ T cells were harvested and re-cultured in media supplemented with IL-2, IL-4, IL-7, IL-15 or IL-21. After 24 hours, cells were analyzed by cytokine bead array, flow cytometry, and mRNA micro-array. Gene networks responsible for specific CD8+ T cell functions were constructed through literature-meta review and publicly available annotation databases. Gene expression data from the experimental groups was imported into this network to visualize the impact of each gc cytokine on the functional polarization of recently-activated CD8+ T cells.ResultsAmong the gc cytokines, IL-2 induced the greatest increase in the expression of co-stimulatory receptors in recently-activated CD8+ T cells. IL-2 increased significantly expression of 4-1BB, GITR, ICOS and OX40, at both the transcriptional and protein level. IL-2 also drove the greatest increase in cellular proliferation and the most robust shift towards a pro-survival phenotype, compared with the other gc cytokines. Both IL-4 and IL-21 enhanced expression of cytotoxic effector proteins, but drove distinct phenotypic polarizations, Th2/Tc2 and NK-like, respectively.ConclusionsOverall, these observations suggest that among gc cytokines, IL-2 may be uniquely capable of synergizing with therapeutic strategies that combine immunization with agonists of co-stimulatory T cell receptors. Previous studies have shown that the timing of IL-2 treatment relative to immunization plays a key role in defining the CD8+ T cell response, and the findings from this study indicate that administration of exogenous IL-2 shortly after the initial antigen-priming event has concluded may augment the receptivity of these cells to subsequent TNFR co-stimulation.

CD16+CD56+ cells are a potential culprit for hematuria in IgA nephropathy.

Hematuria is the first manifestation of urinary abnormality in immunoglobulin A nephropathy (IgAN). Hematuria has recently been reported as a risk factor for deterioration of renal function; however, its cause remains unknown. We analyzed the surface marker of peripheral blood mononuclear cells before and immediately after tonsillectomy in IgAN patients and controls (chronic tonsillitis or tonsillar hypertrophy) by flow cytometry and investigated the association with hematuria. To prove our hypothesis that NK cells induce hematuria, we administered IL-12, activator of NK cells, to HIGA mice. In addition, we transferred cultured NK cells to nude rats and transferred the CD16(+)CD56(+) cells, including NK cells, that are derived from the peripheral blood of IgAN patients immediately after tonsillectomy to nude rats to assess the hematuria level and renal histology of the recipients. We also performed cytotoxicity assays against glomerular endothelial cells by NK cells. We found that IgAN patients who showed rapid deterioration of hematuria after tonsillectomy also displayed a significant increase in CD16(+)CD56(+) cells in the peripheral blood immediately after tonsillectomy. Exogenous administration of IL-12 to HIGA mice induced hematuria. Adoptive transfer of either cells of an NK cell line, or of CD16(+)CD56(+) cells derived from IgAN patients, into nude rats induced hematuria in the recipients. In vitro analysis showed that NK cells exert cytotoxic activity toward human glomerular endothelial cells in a dose-dependent manner. CD16(+)CD56(+) cells seem to be responsible for hematuria in IgAN.

Common gamma chain (¿c) cytokines differentially potentiate TNFR family signaling in antigen-activated CD8 + T cells

BackgroundSeveral members of the common gamma chain (gc) cytokine family are already approved (IL-2) or actively being developed as vaccine adjuvants and cancer immunotherapies. Studies have indicated that co-administration of gc cytokines may enhance the efficacy of immunotherapies that function via direct activation of co-stimulatory T cell receptors. To define the specific influence of gc cytokines on the co-stimulatory capacity of CD8+ T cells and identify combinations with synergistic potential, we investigated the direct impact of gc cytokines on the differentiation and transcriptional profile of recently antigen-primed CD8+ T cells.MethodsNaïve CD8+ T cells were activated with peptide-pulsed APCs. After 48 hours, CD8+ T cells were harvested and re-cultured in media supplemented with IL-2, IL-4, IL-7, IL-15 or IL-21. After 24 hours, cells were analyzed by cytokine bead array, flow cytometry, and mRNA micro-array. Gene networks responsible for specific CD8+ T cell functions were constructed through literature-meta review and publicly available annotation databases. Gene expression data from the experimental groups was imported into this network to visualize the impact of each gc cytokine on the functional polarization of recently-activated CD8+ T cells.ResultsAmong the gc cytokines, IL-2 induced the greatest increase in the expression of co-stimulatory receptors in recently-activated CD8+ T cells. IL-2 increased significantly expression of 4-1BB, GITR, ICOS and OX40, at both the transcriptional and protein level. IL-2 also drove the greatest increase in cellular proliferation and the most robust shift towards a pro-survival phenotype, compared with the other gc cytokines. Both IL-4 and IL-21 enhanced expression of cytotoxic effector proteins, but drove distinct phenotypic polarizations, Th2/Tc2 and NK-like, respectively.ConclusionsOverall, these observations suggest that among gc cytokines, IL-2 may be uniquely capable of synergizing with therapeutic strategies that combine immunization with agonists of co-stimulatory T cell receptors. Previous studies have shown that the timing of IL-2 treatment relative to immunization plays a key role in defining the CD8+ T cell response, and the findings from this study indicate that administration of exogenous IL-2 shortly after the initial antigen-priming event has concluded may augment the receptivity of these cells to subsequent TNFR co-stimulation.

IL-17A and IL-2-Expanded Regulatory T Cells Cooperate to Inhibit Th1-Mediated Rejection of MHC II Disparate Skin Grafts

Several evidences suggest that regulatory T cells (Treg) promote Th17 differentiation. Based on this hypothesis, we tested the effect of IL-17A neutralization in a model of skin transplantation in which long-term graft survival depends on a strong in vivo Treg expansion induced by transient exogenous IL-2 administration. As expected, IL-2 supplementation prevented rejection of MHC class II disparate skin allografts but, surprisingly, not in IL-17A-deficient recipients. We attested that IL-17A was not required for IL-2-mediated Treg expansion, intragraft recruitment or suppressive capacities. Instead, IL-17A prevented allograft rejection by inhibiting Th1 alloreactivity independently of Tregs. Indeed, T-bet expression of naive alloreactive CD4+ T cells and the subsequent Th1 immune response was significantly enhanced in IL-17A deficient mice. Our results illustrate for the first time a protective role of IL-17A in CD4+-mediated allograft rejection process.