Single-cell secretion analysis reveals a dual role for IL-10 in restraining and resolving the TLR4-induced inflammatory response.

Following Toll-like receptor 4 (TLR4) stimulation of macrophages, negative feedback mediated by the anti-inflammatory cytokine interleukin-10 (IL-10) limits the inflammatory response. However, extensive cell-to-cell variability in TLR4-stimulated cytokine secretion raises questions about how negative feedback is robustly implemented. To explore this, we characterize the TLR4-stimulated secretion program in primary murine macrophages using a single-cell microwell assay that enables evaluation of functional autocrine IL-10 signaling. High-dimensional analysis of single-cell data reveals three tiers of TLR4-induced proinflammatory activation based on levels of cytokine secretion. Surprisingly, while IL-10 inhibits TLR4-induced activation in the highest tier, it also contributes to the TLR4-induced activation threshold by regulating which cells transition from non-secreting to secreting states. This role for IL-10 in restraining TLR4 inflammatory activation is largely mediated by intermediate interferon (IFN)-ß signaling, while TNF likely mediates response resolution by IL-10. Thus, cell-to-cell variability in cytokine regulatory motifs provides a means to tailor the TLR4-induced inflammatory response.

IL-10 receptor blockade controls the in vitro infectivity of Leishmania infantum and promotes a Th1 activation in PBMC of dogs with visceral leishmaniasis.

An important strategy to reduce the risk of visceral leishmaniasis (VL) in humans is to control the infection and disease progression in dogs, the domestic reservoir of Leishmania infantum parasites. Certain therapeutic strategies that modulate the host immune response show great potential for the treatment of experimental VL, restoring the impaired effector functions or decreasing host excessive responses. It is known that the overproduction of interleukin-10 (IL-10) promotes parasite replication and disease progression in human VL as well as in canine visceral leishmaniasis (CVL). Thus, in the present study we investigated the potential of the anti-canine IL-10 receptor-blocking monoclonal antibody (Bloq IL-10R) to control and reduce in vitro infectivity of L. infantum and improve the ability of PBMC isolated from VL dogs to alter the lymphoproliferative response and intracytoplasmic cytokines. Overall, GFP+Leishmania showed lower capacity of in vitro infectivity in the presence of Bloq IL-10R. Moreover, addition of Bloq IL-10R in cultured PBMC enhanced T-CD4 and CD8 proliferative response and altered the intracytoplasmic cytokine synthesis, reducing CD4+IL-4+ cells and increasing CD8+IFN-γ+ cells after specific antigen stimulation in PBMC of dogs. Furthermore, we observed an increase of TNF-α levels in supernatant of cultured PBMC under IL-10R neutralizing conditions. Together, our findings are encouraging and reaffirm an important factor that could influence the effectiveness of immune modulation in dogs with VL and suggest that blocking IL-10R activity has the potential to be a useful approach to CVL treatment.

Mucosal IL-10 and IL-10 receptor expression patterns in paediatric patients with ulcerative colitis.

Interleukin-10 (IL-10) is a key anti-inflammatory cytokine. We aimed to assess IL-10 and IL-10 receptor (IL-10R) expression in the gut, and determine whether these patterns are altered in patients with ulcerative colitis (UC). Formalin-fixed paraffin-embedded rectal and transverse colon sections were collected from three groups of patients: (a) control subjects with normal colonoscopy and without history of inflammatory bowel disease; (b) UC patients with extensive colitis or pancolitis (E3/E4 phenotype); and (c) UC patients with limited distal disease (E1/E2 phenotype; n = 8-10 subjects per group). Immunohistochemistry (IHC) was performed to assess expression patterns of IL-10, IL-10R1 and IL-10R2, and was correlated with clinical, endoscopic and histologic severity indices among patients. A trend towards increased IL-10 expression was noted in rectal biopsies of patients with active UC, compared with controls. Moreover, IL-10 levels were significantly increased in transverse colon biopsies of patients with extensive/pancolitis, compared with control subjects and patients with limited distal disease. Rectal IL-10R1 and IL-10R2 levels were comparable between control subject and patients with active UC. However, transverse colon IL-10R1 levels were significantly higher in patients with E3/E4 colitis, compared with controls. Finally, we found no correlation between clinical, endoscopic and histologic severity of inflammation among UC patients and IL-10, IL-10R1 or IL-10R2 expression in rectal sections. Mucosal expression patterns of IL-10 and IL-10R, evaluated by IHC, were overall similar between control subjects and patients with active UC. Given IL-10's anti-inflammatory properties, additional studies are required to determine whether signalling through the IL-10R is altered among these patients.

IL-23-producing IL-10Rα-deficient gut macrophages elicit an IL-22-driven proinflammatory epithelial cell response.

Cytokines maintain intestinal health, but precise intercellular communication networks remain poorly understood. Macrophages are immune sentinels of the intestinal tissue and are critical for gut homeostasis. Here, we show that in a murine inflammatory bowel disease (IBD) model based on macrophage-restricted interleukin-10 (IL-10) receptor deficiency (Cx3cr1Cre:Il10rafl/fl mice), proinflammatory mutant gut macrophages cause severe spontaneous colitis resembling the condition observed in children carrying IL-10R mutations. We establish macrophage-derived IL-23 as the driving factor of this pathology. Specifically, we report that Cx3cr1Cre:Il10rafl/fl:Il23afl/fl mice harboring macrophages deficient for both IL-10R and IL-23 are protected from colitis. By analyzing the epithelial response to proinflammatory macrophages, we provide evidence that T cells of colitic animals produce IL-22, which induces epithelial chemokine expression and detrimental neutrophil recruitment. Collectively, we define macrophage-specific contributions to the induction and pathogenesis of colitis, as manifested in mice harboring IL-10R deficiencies and human IBDs.

Experimental colitis in IL-10-deficient mice ameliorates in the absence of PTPN22.

Interleukin (IL)-10 plays a key role in controlling intestinal inflammation. IL-10-deficient mice and patients with mutations in IL-10 or its receptor, IL-10R, show increased susceptibility to inflammatory bowel diseases (IBD). Protein tyrosine phosphatase, non-receptor type 22 (PTPN22) controls immune cell activation and the equilibrium between regulatory and effector T cells, playing an important role in controlling immune homoeostasis of the gut. Here, we examined the role of PTPN22 in intestinal inflammation of IL-10-deficient (IL-10-/- ) mice. We crossed IL-10-/- mice with PTPN22-/- mice to generate PTPN22-/- IL-10-/- double knock-out mice and induced colitis with dextran sodium sulphate (DSS). In line with previous reports, DSS-induced acute and chronic colitis was exacerbated in IL-10-/- mice compared to wild-type (WT) controls. However, PTPN22-/- IL-10-/- double knock-out mice developed milder disease compared to IL-10-/- mice. IL-17-promoting innate cytokines and T helper type 17 (Th17) cells were markedly increased in PTPN22-/- IL-10-/- mice, but did not provide a protctive function. CXCL1/KC was also increased in PTPN22-/- IL-10-/- mice, but therapeutic injection of CXCL1/KC in IL-10-/- mice did not ameliorate colitis. These results show that PTPN22 promotes intestinal inflammation in IL-10-deficient mice, suggesting that therapeutic targeting of PTPN22 might be beneficial in patients with IBD and mutations in IL-10 and IL-10R.

Blocking IL-10 receptor signaling ameliorates Mycobacterium tuberculosis infection during influenza-induced exacerbation.

Epidemiological findings indicate that coinfection with influenza viruses is associated with an increased risk of death in patients suffering from tuberculosis but the underlying pathomechanisms are not well understood. In this study, we demonstrate that influenza A virus (IAV) coinfection rapidly impairs control of Mycobacterium tuberculosis (Mtb) in C57BL/6 mice. IAV coinfection was associated with significantly increased bacterial loads, reduced survival and a substantial modulation of innate and adaptive immune defenses including an impaired onset and development of Mtb-specific CD4+ T cell responses and the accumulation of macrophages with increased arginase-1 production in the lungs. Our findings strongly indicate that IAV coinfection compromises the host's ability to control Mtb infection via the production of IL-10 which was rapidly induced upon viral infection. The blockade of IL-10 receptor signaling reduced the bacterial load in coinfected mice to a level comparable with that in Mtb-only-infected animals. Taken together, our data suggest that IL-10 signaling constitutes a major pathway that enhances susceptibility to Mtb during concurrent IAV infection.

Comparative proteomic study reveals the enhanced immune response with the blockade of interleukin 10 with anti-IL-10 and anti-IL-10 receptor antibodies in human U937 cells.

Blocking cytokine interleukin 10 (IL-10) at the time of immunisation enhances vaccine induced T cell responses and improves control of tumour cell growth in vivo. However, the effect of an IL-10 blockade on the biological function of macrophages has not been explored. In the current paper, a macrophage precursor cell line, U937 cells, was selected to investigate the differential expression of proteins and relevant cell signalling pathway changes, when stimulated with lipopolysaccharide (LPS) in the presence of antibodies to IL-10 or IL-10 receptor. We used a quantitative proteomic strategy to investigate variations in protein profiles of U937 cells following the treatments with LPS, LPS plus human anti-IL10 antibody and anti-IL10R antibody in 24hrs, respectively. The LPS treatment significantly activated actin-related cell matrix formation and immune response pathways. The addition of anti-IL10 and anti-IL10R antibody further promoted the immune response and potentially effect macrophage survival through PI3K/AKT signalling; however, the latter appeared to also upregulated oncogene XRCC5 and Cajal body associated processes.

Expression of a soluble IL-10 receptor enhances the therapeutic effects of a papillomavirus-associated antitumor vaccine in a murine model.

The presence of IL-10, produced either by tumor cells or immunosuppressive cells, is frequently associated with a poor prognosis for cancer progression. It may also negatively impact anticancer treatments, such as immunotherapies, that otherwise would promote the activation of cytotoxic T cells capable of detecting and destroying malignant cells. In the present study, we evaluated a new adjuvant approach for anticancer immunotherapy using a plasmid vector encoding a soluble form of the IL-10 receptor (pIL-10R). pIL-10R was coadministered to mice with a DNA vaccine encoding the type 16 human papillomavirus (HPV-16) E7 oncoprotein genetically fused with glycoprotein D of herpes simplex virus (HSV) (pgDE7h). Immunization regimens based on the coadministration of pIL-10R and pgDE7h enhanced the antitumor immunity elicited in mice injected with TC-1 cells, which express HPV-16 oncoproteins. The administration of the DNA vaccines by in vivo electroporation further enhanced the anticancer effects of the vaccines, leading to the activation of tumor-infiltrating polyfunctional E7-specific cytotoxic CD8+ T cells and control of the expansion of immunosuppressive cells. In addition, the combination of immunotherapy and pIL-10R allowed the control of tumors in more advanced growth stages that otherwise would not be treatable by the pgDE7h vaccine. In conclusion, the proposed treatment involving the expression of IL-10R enhanced the antitumor protective immunity induced by pgDE7h administration and may contribute to the development of more efficient clinical interventions against HPV-induced tumors.

IL-10 signaling in dendritic cells is required for tolerance induction in a murine model of allergic airway inflammation.

Allergen specific tolerance induction efficiently ameliorates subsequent allergen induced inflammatory responses. The underlying regulatory mechanisms have been attributed mainly to interleukin (IL)-10 produced by diverse hematopoietic cells, while targets of IL-10 in allergen specific tolerance induction have not yet been well defined. Here, we investigate potential cellular targets of IL-10 in allergen specific tolerance induction using mice with a cell type specific inactivation of the IL-10 receptor gene. Allergic airway inflammation was effectively prevented by tolerance induction in mice with IL-10 receptor (IL-10R) deficiency in T or B cells. Similarly, IL-10R on monocytes/macrophages and/or neutrophils was not required for tolerance induction. In contrast, tolerance induction was impaired in mice that lack IL-10R on dendritic cells: those mice developed an allergic response characterized by a pronounced neutrophilic lung infiltration, which was not ameliorated by tolerogenic treatment. In conclusion, our results show that allergen specific tolerance can be effectively induced without a direct impact of IL-10 on cells of the adaptive immune system, and highlight dendritic cells, but not macrophages nor neutrophils, as the main target of IL-10 during tolerance induction.

Targeting cytokines secreted by CD4+ CD25high CD127low regulatory T cells inhibits ovarian cancer progression.

Epithelial ovarian cancer (EOC) is one of the major malignant cancers with high rates of early metastasis in which regulatory T cells (Tregs) play an important role. Tregs suppress immune responses and promote the development of tumours in patients with EOC. However, the underlying mechanisms remain unclear. In this study, we found higher levels of CD4+ CD25high CD127low Tregs in patients with EOC than in patients with benign ovarian tumours and healthy donors. The immune inhibitory effect of Tregs functions by maintaining high levels of immunosuppressive cytokines in EOC. The high levels of Tregs and related cytokines (TGF-ß1 or IL-10) were associated with lymphatic metastasis and FIGO stages of patients with EOC. Expression of matrix metalloproteinase (MMP)-2 and tissue inhibitors of metalloproteinase (TIMP)-2 in EOC cell lines were significantly regulated in the coculture experiment with CD4+ CD25high CD127low Tregs sorted from EOC patients. Levels of MMP-2 and TIMP-2 conversely changed after blocking IL-10R and TGF-ß1R in EOC cells. The invasion ability of EOC cells was also significantly downregulated in this process. The metastasis of EOC cells was correlated with the levels of TGF-ß1 or IL-10. These findings suggested that immunosuppressive cytokines secreted by CD4+ Tregs could be a novel target for inhibiting EOC progression.

Tregopathies: Monogenic diseases resulting in regulatory T-cell deficiency.

Monogenic diseases of the immune system, also known as inborn errors of immunity, are caused by single-gene mutations resulting in immune deficiency and dysregulation. More than 350 diseases have been described to date, and the number is rapidly expanding, with increasing availability of next-generation sequencing facilitating the diagnosis. The spectrum of immune dysregulation is wide, encompassing deficiencies in humoral, cellular, innate, and adaptive immunity; phagocytosis; and the complement system, which lead to autoinflammation and autoimmunity. Multiorgan autoimmunity is a dominant symptom when genetic mutations lead to defects in molecules essential for the development, survival, and/or function of regulatory T (Treg) cells. Studies of "Tregopathies" are providing critical mechanistic information on Treg cell biology, the role of Treg cell-associated molecules, and regulation of peripheral tolerance in human subjects. The pathogenic immune networks underlying these diseases need to be dissected to apply and develop immunomodulatory treatments and design curative treatments using cell and gene therapy. Here we review the pathogenetic mechanisms, clinical presentation, diagnosis, and current and future treatments of major known Tregopathies caused by mutations in FOXP3, CD25, cytotoxic T lymphocyte-associated antigen 4 (CTLA4), LPS-responsive and beige-like anchor protein (LRBA), and BTB domain and CNC homolog 2 (BACH2) and gain-of-function mutations in signal transducer and activator of transcription 3 (STAT3). We also discuss deficiencies in genes encoding STAT5b and IL-10 or IL-10 receptor as potential Tregopathies.

Re-evaluation of IL-10 signaling reveals novel insights on the contribution of the intracellular domain of the IL-10R2 chain.

Interleukin-10 (IL-10) is an anti-inflammatory cytokine that plays a key role in maintaining immune homeostasis. IL-10-mediated responses are triggered upon binding to a heterodimeric receptor complex consisting of IL-10 receptor (IL-10R)1 and IL-10R2. Engagement of the IL-10R complex activates the intracellular kinases Jak1 and Tyk2, but the exact roles of IL-10R2 and IL-10R2-associated signaling via Tyk2 remain unclear. To elucidate the contribution of IL-10R2 and its signaling to IL-10 activity, we re-evaluated IL-10-mediated responses on bone marrow-derived dendritic cells, macrophages and mast cells. By using bone marrow from IL-10R-/- mice it was revealed that IL-10-mediated responses depend on both IL-10R1 and IL-10R2 in all three cell types. On the contrary, bone marrow-derived cells from Tyk2-/- mice showed similar responses to IL-10 as wild-type cells, indicating that signaling via this IL-10R2-associated kinase only plays a limited role. Tyk2 was shown to control the amplitude of STAT3 activation and the up-regulation of downstream SOCS3 expression. SOCS3 up-regulation was found to be cell-type dependent and correlated with the lack of early suppression of LPS-induced TNF-α in dendritic cells. Further investigation of the IL-10R complex revealed that both the extracellular and intracellular domains of IL-10R2 influence the conformation of IL-10R1 and that both domains were required for transducing IL-10 signals. This observation highlights a novel role for the intracellular domain of IL-10R2 in the molecular mechanisms of IL-10R activation.

Induction of Live Cell Phagocytosis by a Specific Combination of Inflammatory Stimuli.

Conditions of severe hyper-inflammation can lead to uncontrolled activation of macrophages, and the ensuing phagocytosis of live cells. However, relationships between inflammatory stimuli and uncontrolled phagocytosis of live cells by macrophages are poorly understood. To identify mediators of this process, we established phagocytosis assays of live cells by stimulating macrophages with CpG DNA, interferon-γ, and anti-interleukin-10 receptor antibody. In this model, various cell surface receptors were upregulated on macrophages, and phagocytosis of live cells was induced in a Rac1-dependent manner. Subsequent inhibition of the ICAM-1, VCAM-1, and both of these receptors abolished in vitro and in vivo phagocytosis of live T cells, myeloid cells, and B cells, respectively. Specifically, the reduction in lymphocyte numbers due to in vivo activation of macrophages was ameliorated in Icam-1-deficient mice. In addition, overexpression of ICAM-1 or VCAM-1 in non-phagocytic NIH3T3 cells led to active phagocytosis of live cells. These data indicate molecular mechanisms underlying live cell phagocytosis induced by hyper-inflammation, and this experimental model will be useful to clarify the pathophysiological mechanisms of hemophagocytosis and to indicate therapeutic targets.

Reversing the polarization of tumor-associated macrophages inhibits tumor metastasis.

OBJECTIVE: The M2 phenotype is dominant in tumor associated macrophages (TAM), and plays a key role in promoting tumor growth, invasion and metastasis. Converting TAM polarization from M2 to M1 may contribute to eliciting anti-tumor-specific immune responses and inhibiting tumor metastasis. In this study, the effect of reversing the polarization of TAM on tumor metastasis was investigated. METHODS: Peritoneal macrophages were obtained from BABL/c mice, and M2 polarization was induced by IL-4. In an in vivo experiment, BABL/c mice were transplanted with 4T1 tumor cells. In vitro and in vivo experimental studies, M2 macrophage polarization was reversed with CpG-DNA or CpG-DNA combined with anti-IL-10R Ab. CD68, MHCII and FRß molecular expression in macrophages were examined with immunofluorescence staining. The mRNA expression of IL-2, IL-6, IL-13, VEGF and MMP-9 were detected with RT-PCR. VEGF and MMP-9 protein expression of tumors in situ was measured by western blot assay. Lung-metastasis of the tumor was observed and assessed by micro-CT. RESULTS: CpG-DNA and CpG-DNA combined with anti-IL-10R Ab could promote MHCII, IL-2, IL-6 and IL-13 molecular expression, and suppress the expression of FRß, MMP-9 and VEGF, in both freshly isolated peritoneal macrophages and M2 macrophages. In the CpG-DNA combined with anti-IL-10R Ab injecting group, the percentage of CD68+ MHCII+ cells were significantly higher than that of CD68+FRß+ cells (P<0.05). This was distinct from the result of the control group, which CD68+ FRß+ was higher than CD68+MHCII+cells (P<0.01). Furthermore, VEGF-A and MMP-9 level in primary tumor tissues in the experimental group was significantly lower (P<0.01), compared to the control group. Moreover, the number of detectable lung-metastasis foci was significantly lower in the experimental group than in the control group (P<0.05). CONCLUSION: Reversing the polarization of TAM from M2 to M1 phenotype can inhibit tumor metastasis.

Tyrosine kinase 2 is not limiting human antiviral type III interferon responses.

Tyrosine kinase 2 (TYK2) associates with interferon (IFN) alpha receptor, IL-10 receptor (IL-10R) beta and other cytokine receptor subunits for signal transduction, in response to various cytokines, including type-I and type-III IFNs, IL-6, IL-10, IL-12 and IL-23. Data on TYK2 dependence on cytokine responses and in vivo consequences of TYK2 deficiency are inconsistent. We investigated a TYK2 deficient patient, presenting with eczema, skin abscesses, respiratory infections and IgE levels >1000 U/mL, without viral or mycobacterial infections and a corresponding cellular model to analyze the role of TYK2 in type-III IFN mediated responses and NK-cell function. We established a novel simple diagnostic monocyte assay to show that the mutation completely abolishes the IFN-α mediated antiviral response. It also partly reduces IL-10 but not IL-6 mediated signaling associated with reduced IL-10Rß expression. However, we found almost normal type-III IFN signaling associated with minimal impairment of virus control in a TYK2 deficient human cell line. Contrary to observations in TYK2 deficient mice, NK-cell phenotype and function, including IL-12/IL-18 mediated responses, were normal in the patient. Thus, preserved type-III IFN responses and normal NK-cell function may contribute to antiviral protection in TYK2 deficiency leading to a surprisingly mild human phenotype.

IL-10 Potentiates Differentiation of Human Induced Regulatory T Cells via STAT3 and Foxo1.

Foxp3(+) regulatory T cells (Tregs) play essential roles in maintaining the immune balance. Although the majority of Tregs are formed in the thymus, increasing evidence suggests that induced Tregs (iTregs) may be generated in the periphery from naive cells. However, unlike in the murine system, significant controversy exists regarding the suppressive capacity of these iTregs in humans, especially those generated in vitro in the presence of TGF-ß. Although it is well known that IL-10 is an important mediator of Treg suppression, the action of IL-10 on Tregs themselves is less well characterized. In this article, we show that the presence of IL-10, in addition to TGF-ß, leads to increased expansion of Foxp3(+) iTregs with enhanced CTLA-4 expression and suppressive capability, comparable to that of natural Tregs. This process is dependent on IL-10R-mediated STAT3 signaling, as supported by the lack of an IL-10 effect in patients with IL-10R deficiency and dominant-negative STAT3 mutation. Additionally, IL-10-induced inhibition of Akt phosphorylation and subsequent preservation of Foxo1 function are critical. These results highlight a previously unrecognized function of IL-10 in human iTreg generation, with potential therapeutic implications for the treatment of immune diseases, such as autoimmunity and allergy.

Kupffer Cells Support Hepatitis B Virus-Mediated CD8+ T Cell Exhaustion via Hepatitis B Core Antigen-TLR2 Interactions in Mice.

Hepatitis B virus (HBV) persistence is a fundamental process in chronic HBV infection and a key factor in all related liver diseases; however, the mechanisms have yet to be elucidated. We studied the role of TLR2 in HBV persistence using a well-established HBV-carrier mouse model generated by hydrodynamically injecting a phospho-adeno-associated virus/HBV1.2 plasmid into mice. We found that a genetic deficiency in TLR2 improves HBV elimination, whereas activating TLR2 led to more stable HBV persistence, suggesting that TLR2 activation is critical in HBV persistence. Furthermore, we noted that TLR2 activation could inhibit CD8(+) T cell function, causing the exhaustion phenotype in HBV-carrier mice, because TLR2 deficiency might rescue CD8(+) T cell function in a cellular adoptive experiment. TLR2 expression on Kupffer cells (KCs) was upregulated in HBV-carrier mice, which accounts for HBV persistence, because the difference in anti-HBV immunity between HBV-carrier wild-type and Tlr2(-/-) mice did not exist after KC depletion. In addition, similar to TLR2 deficiency, after KC depletion, CD8(+) T cells were more efficiently activated in HBV-carrier mice, leading to rapid HBV elimination. KCs produced more IL-10 upon TLR2 activation in response to direct hepatitis B core Ag stimulation, and the elevated IL-10 inhibited CD8(+) T cell function in HBV-carrier mice, because IL-10 deficiency or anti-IL-10R treatment resulted in CD8(+) T cells with stronger antiviral function. In conclusion, KCs support liver tolerance by inducing anti-HBV CD8(+) T cell exhaustion via IL-10 production after TLR2 activation by hepatitis B core Ag stimulation.

The paradoxical role of IL-10 in immunity and cancer.

Interleukin-10 (IL-10) produced by a wide-variety of cells is a highly pleiotropic cytokine. It has been implicated in the pathogenesis and/or development of autoimmune diseases and cancer, although it displays differential effects that seem to be contradictory sometimes. The ultimate role of this cytokine in disease, however, cannot be fully determined until the immunological contexts that regulate its function are further elucidated. In this review, we will discuss a wide variety of evidence of IL-10 in immunity and cancer in an effort to illuminate the remaining mysteries in the function of this cytokine that, when fully understood, may prove to be a powerful tool in the battle against cancer.

Depletion of tumor-associated macrophages enhances the anti-tumor immunity induced by a Toll-like receptor agonist-conjugated peptide.

It has been reported that lipopeptides can be used to elicit cytotoxic T lymphocyte (CTL) responses against viral diseases and cancer. In our previous study, we determined that mono-palmitoylated peptides can enhance anti-tumor responses in the absence of adjuvant activity. To investigate whether di-palmitoylated peptides with TLR2 agonist activity are able to induce anti-tumor immunity, we synthesized a di-palmitic acid-conjugated long peptide that contains a murine CTL epitope of HPV E749-57 (Pam2IDG). Pam2IDG stimulated the maturation of bone marrow-derived dendritic cells (BMDCs) through TLR2/6. After immunization, Pam2IDG induced higher levels of T cell responses than those obtained with its non-lipidated counterpart (IDG). In the prophylactic model, Pam2IDG immunization completely inhibited tumor growth, whereas IDG immunization was unable to inhibit tumor growth. However, Pam2IDG immunization could not effectively inhibit the growth of established tumors. Therefore, we further investigated whether the depletion of immunosuppressive factors could improve the therapeutic effects of Pam2IDG. Our data indicate that treatment with Pam2IDG combined with clodronate/liposome delays tumor growth and increases the survival rate. We also observed that the therapeutic effects of Pam2IDG are improved by diminishing the function of tumor-associate macrophages (TAMs) and through the use of an IL10 receptor blocking antibody or a Cyclooxygenase 2 (Cox-2) inhibitor. In conclusion, the depletion of TAMs may enhance the anti-tumor immunity of a TLR2 agonist-conjugated peptide.

IL-10 signalling blockade at the time of immunization inhibits Human papillomavirus 16 E7 transformed TC-1 tumour cells growth in mice.

IL-10 signalling blockade by intra-peritoneal injection of anti-IL-10 receptor antibodies at the time of immunization enhances vaccine induced CD8+ T cell responses and promotes bacteria, parasitic and viral control. We now show that blockade of IL-10 signalling at the time of immunization enhances vaccine induced antigen specific CD8+ T cell responses to both dominant and subdominant CTL epitopes. Injection of anti-IL-10 receptor antibodies subcutaneous at the time of immunization also enhances CD8+ T cell responses. Furthermore, IL-10 signalling blockade at the time of a Human papillomavirus 16 E7 peptide/LPS immunization, prevents HPV16 E7 transformed TC-1 tumour growth in mice. Immunization in the presence of anti-IL-10R antibodies and Monophosphoryl lipid A, generates antigen specific CD8+ T cell responses similar to immunization with LPS. Our results suggest that immunization and IL-10 signalling blockade may provide a novel way for the development of therapeutic vaccines against cancer.