IFN-γ Expression In CD8 Research Articles

Abstract 162: Fatty acid synthesis blockade synergizes with immune checkpoint blockade by repolarizing macrophages and enhancing anti-tumor cytotoxicity in gastric cancer

Abstract Background: Immune checkpoint blockade (ICB), which unleashes potent CD8+ T cell-based anti-tumor responses, has emerged as a promising therapeutic option for several cancer types. However, response rates stay limited in gastric cancer (GC) patients, indicating the necessity of combinational strategies to overcome other immunosuppressive factors. Fatty acid synthesis (FAS) is an indispensable metabolic pathway proven to support cancer cell survival. The rate-limiting enzyme of FAS, FASN, is upregulated in several cancers and could contribute to cancer immune escape. Though some investigations propose that specific gene alterations can cause FASN upregulation under the GC setting, the immunoregulatory role and therapeutic value of FASN in the tumor microenvironment (TME) of GC have not been fully elucidated. Methods: FASN expression was measured by Western Blotting and flow cytometry. Bioinformatic analysis was conducted via an online website (www.camoip.net). The syngeneic GC mouse model was established by subcutaneous inoculation of MFC cells in 615-line mice, treated by vehicles, FASN inhibitor (FASNi), anti-PD-1 antibodies, or FASNi+anti-PD-1 antibodies. TAM and CD8+ T cell depletion was implemented by anti-CSF1R and anti-CD8α antibodies. Bone marrow-derived macrophages (BMDMs) were extracted from the femurs of 615-line mice, induced by M-CSF and stimulated by LPS or IL-4. The proportions and functional status of immune cells were identified by flow cytometry. Results: FASN was upregulated in GC tissues and GC human cell lines. Bioinformatic analyses demonstrated that FASN expression was related to poor infiltration of cytotoxic lymphocytes, as well as the downregulation of pathways related to anti-cancer immune responses. FASNi inhibited tumor growth in a syngeneic GC mouse model, with elevated PD-1 and IFN-γ expression in CD8+ T cells and increased NK cell infiltration, suggesting the enhancement in anti-cancer cytotoxicity. Meanwhile, FASNi induced a shift from M2-like to M1-like in tumor-associated macrophages (TAM). We detected that M2-like TAM and CD8+ T cells harbored higher FASN expression than other immune cells in GC mouse tissues, and tumor-suppressing effects of FASN inhibition could be reversed by TAM and CD8+ T cell depletion, which corroborated the key role of TAM and CD8+ T cells in shaping FASN-mediated tumor regulation. FASNi could also repolarize BMDMs from M1-like to M2-like in vitro. Finally, FASNi exhibited synergistic tumor control with anti-PD-1 therapy in GC and melanoma mouse models. Conclusion: This work sheds light on the impact of FASN on TME modulation of GC. We proved that FASNi could repolarize M2-like TAM to M1 phenotypes and promote cytotoxic lymphocyte-mediated anti-tumor immunity. Our results propose FASN inhibition as a potential therapeutic strategy to sensitize ICB therapy in GC. Citation Format: Hanbing Wang, Tao Shi, Yue Wang, Yuting Luo, Yunfeng Pan, Xiaoyu Zhou, Yue Zhang, Baorui Liu, Jia Wei. Fatty acid synthesis blockade synergizes with immune checkpoint blockade by repolarizing macrophages and enhancing anti-tumor cytotoxicity in gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 162.

CD4+ Th1 and Th17 responses and multifunctional CD8 T lymphocytes associated with cure or disease worsening in human visceral leishmaniasis.

In VL, a proinflammatory phenotype is typically associated with enhanced phagocytosis and a Th1 mediated immune response resulting in infection control. In contrast, an anti-inflammatory phenotype, associated with a predominant regulatory response, typically enables intracellular multiplication of Leishmania parasites and disease progression. To investigate the impact of chemotherapy on Th2 and Th17 immune responses in patients with visceral leishmaniasis (VL), we assessed all combinations of intracellular expression of IFN-γ, IL-10, IL-4 and IL-17 in the CD4+ and CD8+ T cell populations of peripheral blood mononuclear cell (PBMC) samples from patients, after antigenic stimulation with Leishmania lysate, throughout treatment and follow-up. As increases in spleen and liver sizes and decreases in hematocrit, hemogloblin, erythrocytes, monocytes, leukocytes and platelets levels are strongly related to the disease, we studied the correlations between the frequencies of T cells producing the afore mentioned cytokines, individually and in combination, and these variables, as markers of disease or cure. We found that the frequency of IFN-γ-producingCD4+ T cells increased until the end of chemotherapy with Glucantime® or AmBisome ®, while IL-10, IL-4 and IL-17-producing CD4+ T cells peaked on day 7 following the start of treatment. Although the frequency of CD4+IL-17+ cells decreased during treatment an increase was observed after clinical cure. The frequency of CD4+ T cells producing only IFN-γ or IL-17 correlated with blood monocytes levels. Frequencies of double-producers of IFN-γ and IL-10 or IL-4 correlated positively with eosinophils and platelets levels. Together, this suggest that IFN-γ drives the immune response towards Th1 at cure. In contrast, and associated with disease or Th2 response, the frequency of CD4+ IL-10+ cells correlated positively with spleen sizes and negatively with circulating monocyte levels, while the frequency of CD4+ producing both IL-4 and IL-10 correlated negatively with platelets levels. The frequency of CD8+ single-producers of IFN-γ increased from day 21 to 90 while that of single-producers of IL-10 peaked on day 7, of IL-4 on day 30 and of IL-17, on day 180. IFN-γ expression in CD8+ single- and double-producers of cytokines was indicative of an immune response associated with cure. In contrast, frequencies of CD8+ double-producers of IL-4 and IL-10, IL-4 and IL-17 and IL-10 and IL-17 and producers of three and four cytokines, were associated with disease and were low after the cure. Frequencies of CD8+ T cells producing IFN-γ alone or with IL-17 were positively correlated with platelets levels. In contrast, as markers of disease: 1) frequencies of single producers of IL-10 correlated negatively with leukocytes levels, 2) frequencies of double producers of IL-4 and IL-10 correlated negatively with platelet, leukocyte, lymphocyte and circulating monocyte levels, 3) frequencies of triple-producers of IFN-γ, IL-4 and IL-10 correlated negatively with platelet, leukocyte and neutrophil levels and 4) frequencies of producers of IFN-γ, IL-4, IL-10 and IL-17 simultaneously correlated positively with spleen size, and negatively with leukocyte and neutrophil levels. Our results confirmed that the clinical improvement of VL patients correlates with the decrease of an IL-4 and IL-10 CD4+Th2 response, the recovery of CD4+ Th1 and Th17 responses and the frequency of CD8+ single-producers of IFN-γ and double producers of IFN-γ and IL-17.

Abstract 6433: A novel bacterial strain, CJRB-101, induces anti-cancer effects by repolarization of M2 to CXCL9 and CXCL10 dual expressing M1 macrophages in humanized non-small cell lung cancer mice models

Abstract Backgrounds: Live biotherapeutic products (LBPs) emerged as potential therapeutics to overcome the limitation of ICIs. This research shows that CJRB-101, a novel bacterial strain, can improve anti-tumor effects in synergy with pembrolizumab in non-small cell lung cancer (NSCLC). Objectives and Methods: Tumors from NSCLC patients (anti-PD-1 refractory and resistant) were transplanted into Hu-CD34-NSG to establish humanized patient-derived xenograft (PDX) mice models. Five models (YHIM-2003, 2004, 2009, 2010 and 2014) were treated with CJRB-101 at low (5 × 107 CFU) or high (109 CFU) doses, or with pembrolizumab (10 mg/kg, i.p., Q5D) or in combination. Tumor growth inhibition (TGI) rate was measured. Tumor microenvironment (TME) was analyzed using multiplex IHC, flow cytometry and single cell RNA sequencing. Ex-vivo assays were performed to validate in silico findings. Results: Tumor in PDX models was unresponsive to pembrolizumab alone, however, in combination with CJRB-101 effectively suppressed tumor growth. The synergy was highlighted in YHIM-2009 where TGI was 10-fold higher (56%) than pembrolizumab group (5%). Immune profiling revealed that macrophages may be responsible for the anti-tumor effects of CJRB-101. IHC showed significantly increased antigen presenting specialized DCs (CD16+CD68−CD11c+) and granzyme B+ CD8+ T cells in the tumor by CJRB-101 compared to pembrolizumab (p<0.01). This suggested that CJRB-101 induced infiltration of cytotoxic CD8 T cells into the tumor nest by enhancing antigen presenting machinery. Trajectory analysis showed that CJRB-101 induced repolarization of M2 to M1 macrophages, characterized by high expression of CXCL9/10. CXCL9+/10+ M1 macrophages were comparatively more abundant in the combination group (23.11%) than the pembrolizumab group (0.91%). CXCL9/CXCL10 expression in macrophages was higher in the CJRB-101 group compared to the pembrolizumab group (p<0.0001). The combination group (10.84%) had a higher relative abundance of CD8+ T cells compared to the pembrolizumab group (1.58%) and higher IFNγ expression in CD8+ T cells compared to the pembrolizumab group (p=0.0152), suggesting that CJRB-101 repolarized macrophages and recruited active CD8+ T cells. Co-culture assays using bone marrow-derived macrophages validated that CJRB-101 drove differentiation towards F4/80+ or MHC II+ expressing M1 macrophage (p<0.0001) and repolarized existing M2 (CD206+) to M1 (p=0.0002). Conclusion: Combination treatment of CJRB-101 with anti-PD-1 showed synergistic anti-tumor effects via repolarization of M2 to M1 macrophages, leading to activation of CD8+ T cells in TME. Especially, CXCL9+/10+ M1 macrophage playing a key role in TGI induced by CJRB-101 in NSCLC models. Findings from this study provided rationale for clinical investigation of CJRB-101. Citation Format: Arim Min, Chun-bong Synn, Seong-san Kang, Bo-eun Kwon, Junwon Yang, Hyunkyung Park, Jieun Im, Hyunjeong Kim, Sujeong Beak, Dong Kwon Kim, Jii Bum Lee, Hyeonseok Oh, Seung Min Yang, Yu Jin Han, Mi hyun Kim, Heekyung Han, Kwangmin Na, Young Taek Kim, Sungwoo Lee, Mi Ran Yun, Jae Hwan Kim, Youngseon Byeon, Young Seob Kim, Ji Yun Lee, Chang Gon Kim, Min Hee Hong, Sun Min Lim, Kyoung-Ho Pyo, Byoung Chul Cho. A novel bacterial strain, CJRB-101, induces anti-cancer effects by repolarization of M2 to CXCL9 and CXCL10 dual expressing M1 macrophages in humanized non-small cell lung cancer mice models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6433.

TGF-β-Induced FLRT3 Attenuation Is Essential for Cancer-Associated Fibroblast-Mediated Epithelial-Mesenchymal Transition in Colorectal Cancer.

CAFs enhance colorectal cancer aggressiveness by reducing FLRT3 expression through activating TGF-β/SMAD4 signaling pathway. CAF-targeted therapy and/or LY2109761 were promising treatments for colorectal cancer.

Interleukin 32 Promotes Foxp3+ Treg Cell Development and CD8+ T Cell Function in Human Esophageal Squamous Cell Carcinoma Microenvironment.

Proinflammatory cytokine interleukin 32 (IL-32) is involved in infectious diseases and cancer, but what subtypes of immune cells express IL-32 and its roles in tumor microenvironment (TME) have not been well discussed. In this study, we applied bioinformatics to analyze single-cell RNA sequencing data about tumor-infiltrating immune cells from esophageal squamous cell carcinoma (ESCC) TME and analyzed IL-32 expression in different immune cell types. We found CD4+ regulatory T cells (Treg cells) express the highest level of IL-32, while proliferating T and natural killer cells expressed relatively lower levels. Knocking down of IL-32 reduced Foxp3 and interferon gamma (IFNγ) expressions in CD4+ and CD8+ T cells, respectively. IL-32 was positively correlated with Foxp3, IFNG, and GZMB expression but was negatively correlated with proliferation score. IL-32 may have a contradictory role in the TME such as it promotes IFNγ expression in CD8+ T cells, which enhances the antitumor activity, but at the same time induces Foxp3 expression in CD4+ T cells, which suppresses the tumor immune response. Our results demonstrate different roles of IL-32 in Treg cells and CD8+ T cells and suggest that it can potentially be a target for ESCC cancer immunosuppressive therapy.

Rapid Depletion of Intratumoral Regulatory T Cells Induces Synchronized CD8 T- and NK-cell Activation and IFNγ-Dependent Tumor Vessel Regression.

Regulatory T cells (Tregs) are known to inhibit antitumor immunity, yet the specific mechanism by which intratumoral Tregs promote tumor growth remains unclear. To better understand the roles of intratumoral Tregs, we selectively depleted tumor-infiltrating Tregs using anti-CD25-F(ab')2 near-infrared photoimmunotherapy. Depletion of tumor-infiltrating Tregs induced transient but synchronized IFNγ expression in CD8 T and natural killer (NK) cells. Despite the small fraction of CD8 T and NK cells contained within examined tumors, IFNγ produced by these CD8 T and NK cells led to efficient and rapid tumor vessel regression, intratumoral ischemia, and tumor necrosis/apoptosis and growth suppression. IFNγ receptor expression on vascular endothelial cells was required for these effects. Similar findings were observed in the early phase of systemic Treg depletion in tumor-bearing Foxp3DTR mice; combination with IL15 therapy further inhibited tumor growth and achieved increased complete regression. These results indicate the pivotal roles of intratumoral Tregs in maintaining tumor vessels and tumor growth by suppressing CD8 T and NK cells from producing IFNγ, providing insight into the mechanism of Treg-targeting therapies. SIGNIFICANCE: Intratumoral Treg depletion induces synchronized intratumoral CD8 T- and NK-cell activation, IFNγ-dependent tumor vessel regression, and ischemic tumor necrosis/apoptosis, indicating the roles of intratumoral Tregs to support the tumor vasculature. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/11/3092/F1.large.jpg.

The Role of T Cells Reactive to the Cathelicidin Antimicrobial Peptide LL-37 in Acute Coronary Syndrome and Plaque Calcification

The human cationic anti-microbial peptide LL-37 is a T cell self-antigen in patients with psoriasis, who have increased risk of cardiovascular events. However, the role of LL-37 as a T cell self-antigen in the context of atherosclerosis remains unclear. The objective of this study was to test for the presence of T cells reactive to LL-37 in patients with acute coronary syndrome (ACS). Furthermore, the role of T cells reactive to LL-37 in atherosclerosis was assessed using apoE−/− mice immunized with the LL-37 mouse ortholog, mCRAMP. Peripheral blood mononuclear cells (PBMCs) from patients with ACS were stimulated with LL-37. PBMCs from stable coronary artery disease (CAD) patients or self-reported subjects served as controls. T cell memory responses were analyzed with flow cytometry. Stimulation of PBMCs with LL-37 reduced CD8+ effector T cell responses in controls and patients with stable CAD but not in ACS and was associated with reduced programmed cell death protein 1 (PDCD1) mRNA expression. For the mouse studies, donor apoE−/− mice were immunized with mCRAMP or adjuvant as controls, then T cells were isolated and adoptively transferred into recipient apoE−/− mice fed a Western diet. Recipient mice were euthanized after 5 weeks. Whole aortas and hearts were collected for analysis of atherosclerotic plaques. Spleens were collected for flow cytometric and mRNA expression analysis. Adoptive transfer experiments in apoE−/− mice showed a 28% reduction in aortic plaque area in mCRAMP T cell recipient mice (P < 0.05). Fifty six percent of adjuvant T cell recipient mice showed calcification in atherosclerotic plaques, compared to none in the mCRAMP T cell recipient mice (Fisher’s exact test P = 0.003). Recipients of T cells from mice immunized with mCRAMP had increased IL-10 and IFN-γ expression in CD8+ T cells compared to controls. In conclusion, the persistence of CD8+ effector T cell response in PBMCs from patients with ACS stimulated with LL-37 suggests that LL-37-reactive T cells may be involved in the acute event. Furthermore, studies in apoE−/− mice suggest that T cells reactive to mCRAMP are functionally active in atherosclerosis and may be involved in modulating plaque calcification.

Flowcytometric Cytokine Profiling of T- Lymphocytes in Pediatric Aplastic Anemia

Background: Aplastic anemia is a result of complex immune mediated marrow injury. As activated T cells play a pivotal role in immune response, study of cytokine profile in patients of aplastic anaemia (AA) will be of use in understanding the initiation and propagation of the disease. Methods: Thirty children with AA between ages 6 months to 18 years were included along with age & sex matched controls. Complete blood count (CBC) and bone marrow examination was done. They were grouped as Very severe aplastic anemia (VSAA), Severe aplastic anemia (SAA) and Non severe aplastic anemia (NSAA) as per standard guidelines. Intracytoplasmic expression of Type 1 cytokine (IFNγ) and Type 2 cytokine (IL4) was determined by Flow cytometry (FCM). CD8 and CD4 cell counts were also determined by FCM. Result: CD8 cell count was higher than the CD4 counts with reversal of CD4:CD8 ratio in all patients. Mean intracytoplasmic IFNγ expression in CD8 and CD4 cells was higher in cases than in controls (p<0.0001) and showed graded positive correlation with the severity of the disease (p<0.0001). Type 2 cytokine (IL4) did not show any such correlation. Conclusion: IFN gamma expression in T cells can be useful in predicting severity of the disease and also response to immunosuppression.

Chronic Hepatitis C Virus Infection Impairs M1 Macrophage Differentiation and Contributes to CD8+ T-Cell Dysfunction.

Chronic hepatitis C virus (HCV) infection causes generalized CD8+ T cell impairment, not limited to HCV-specific CD8+ T-cells. Liver-infiltrating monocyte-derived macrophages (MDMs) contribute to the local micro-environment and can interact with and influence cells routinely trafficking through the liver, including CD8+ T-cells. MDMs can be polarized into M1 (classically activated) and M2a, M2b, and M2c (alternatively activated) phenotypes that perform pro- and anti-inflammatory functions, respectively. The impact of chronic HCV infection on MDM subset functions is not known. Our results show that M1 cells generated from chronic HCV patients acquire M2 characteristics, such as increased CD86 expression and IL-10 secretion, compared to uninfected controls. In contrast, M2 subsets from HCV-infected individuals acquired M1-like features by secreting more IL-12 and IFN-γ. The severity of liver disease was also associated with altered macrophage subset differentiation. In co-cultures with autologous CD8+ T-cells from controls, M1 macrophages alone significantly increased CD8+ T cell IFN-γ expression in a cytokine-independent and cell-contact-dependent manner. However, M1 macrophages from HCV-infected individuals significantly decreased IFN-γ expression in CD8+ T-cells. Therefore, altered M1 macrophage differentiation in chronic HCV infection may contribute to observed CD8+ T-cell dysfunction. Understanding the immunological perturbations in chronic HCV infection will lead to the identification of therapeutic targets to restore immune function in HCV+ individuals, and aid in the mitigation of associated negative clinical outcomes.

Humoral and cellular immune response of mice challenged with Yersinia pestis antigenic preparations

ObjectivesThe plague, which is an infectious disease caused by Yersinia pestis, still threatens many populations in several countries. The worldwide increase in human plague cases and the potential use of the bacteria as a biological weapon reinforce the need to study the immunity that is induced by potential vaccine candidates. To determine the immunogenicity of antigenic preparations based on the F1 protein and the total extract from Y. pestis, we assessed the role of these antigens in inducing an immune response. MethodsThe immunogenicity of antigenic preparations based on the Y. pestis (YP) total extract and the Y. pestis fraction 1 capsular antigen protein (F1) was determined in Swiss-Webster mice immunized with 40μg or 20μg for each preparation. Immunophenotyping was performed by flow cytometry. ResultsAnimals immunized with the YP total extract did not elicit detectable anti-F1 antibodies (Ab) in the hemaglutination/inhibition (HA/HI) test. Animals immunized with 40μg or 20μg of the F1 protein produced anti-F1 Abs, with titres ranging from 1/16 to 1/8132. The average of CD3+–CD4+ and CD3+–CD8+ T cells did not differ significantly between the groups. Neither YP total extract nor F1 protein induced a significant expression of IFN-γ and IL-10 in CD4+ T lymphocytes. In addition, F1 failed to induce IFN-γ expression in CD8+ T cells, unlike the YP total extract. ConclusionThe results showed that F1 protein is not an immunogenic T cell antigen, although the YP total extract (40μg dose) favoured CD8+ T cell-mediated cellular immunity.

Adjuvant-active aqueous extracts from Artemisia rupestris L. improve immune responses through TLR4 signaling pathway

Activating innate immunity by an adjuvant is required in vaccine development. The study aims to investigate adjuvant effects of aqueous extracts of Artemisia rupestris L. (AEAR) in vivo and in vitro. ICR mice were subcutaneously administered with antigen and AEAR at various doses to evaluate their immune responses of antibodies, dendritic cells (DCs), regulatory T cells (Treg), splenic lymphocyte, and cytokine. The evaluation results showed that AEAR could largely increase titers of antigen-specific antibodies (IgG, IgG1, and IgG2a) and T cell proliferation. AEAR also increased expression of IFN-γ in CD8+T cells as well as IL-4 and INF-γ expression in CD4+T cells. Expression levels of MHC-II, CD40, CD80, and CD86 on DCs were significantly elevated, whereas the Treg frequency was significantly decreased. AEAR (200μg) showed remarkable adjuvant activity. Furthermore, AEAR enhanced MHC-II, CD40, CD80, and CD86 expression as well as the yields of TNF-α and IL-12 on DCs through toll-like receptor4 (TLR4) in vitro. Those results indicated that AEAR could serve as an efficacious immune stimulator for vaccines because it significantly enhanced specific immune responses by promoting DCs maturation and reduced Treg through TLR4 signaling pathway.

FATS deficiency in mice limits melanoma progression via converting the tumor immune microenvironment to an anti-tumor state

Abstract C10orf90 is a newly identified common fragile sites gene named fragile-site associated tumor suppressor (FATS) according its tumor suppressive function in FATS overexpressive cancer cell lines. However, our FATS knockout mice data suggested that FATS may service as an important immune regulator. In this study, we found that FATS deficiency in mice can significantly inhibit the tumor incidence and growth of melanoma implanted with B16 cells. In addition, FATS deficient mice obviously increased the numbers and frequency of tumor suppressor cells CD3+ T cell, γδT and NK cells, and decreased tumor promoter cells PMN-MDSC and Treg cell, in tumor tissues. Notably, DCs which play a key role in immune response initiation were dramatically increased in tumor tissues of the FATS deficiency mice. Furthermore, IL-2 which is crucial to the activation of T cells, significantly increased in serum of the FATS deficiency mice compared with the WT mice, meanwhile, other cytokines, such as IFN-γ and IL-12 were increased as well. Importantly, FATS deficiency increased the expression of IFN-γ in CD8+ T cells and CD4+ T cells and facilitated the activation of these T cells separated from the mice tumor tissues. Also, perforin and granzyme, the pivotal effector molecules in tumor cytotoxicity, raised in the CD8+ T cells from the melanoma tissues of FATS deficiency mice. Consistently, in the ex vitro study, DC separated from FATS deficiency mice with melanoma could significantly promote the proliferation and activation of T cells. These results demonstrate the inhibitory effect of FATS deficiency on the progression of melanoma via regulating tumor immune microenvironment and suggest that FATS could be a potential immune therapeutic target for malignant tumors.

Chuanminshen violaceum polysaccharides improve the immune responses of foot-and-mouth disease vaccine in mice

Water-soluble polysaccharides from Chuanminshen violaceum (CVPS) were evaluated for their physicochemical properties, monosaccharide composition, and adjuvant potential to specific cellular and humoral immune responses in a mouse model of foot-and-mouth disease virus (FMDV) vaccination. The average molecular weight (Mw) of the CVPS was 968.31kDa. The monosaccharide components of the CVPS was rhamnose, arabinose, fucose, mannose, glucose, and galactose with a relative mass of 6.29%, 21.87%, 16.59%, 12.54%, 13.07%, and 28.05%, respectively. Administering CVPS as an adjuvant significantly enhanced the phagocytic capacity of peritoneal macrophages, splenocyte proliferation, and the activity of NK cells and CTL as well as increased FMDV-specific IgG and IgG subclass antibody titers. Moreover, CVPS increased the expression of IL-2, IFN-γ, and IL-4 in CD4+ T cells and IFN-γ expression in CD8+ T cells. Additionally, CVPS enhanced CD40+, CD80+, and CD86+ expression on DCs. Moreover, CVPS upregulated MHC-I/II, TLR-2/4 mRNA levels. In contrast, CVPS downregulated TGF-β mRNA expression and the frequency of CD4+CD25+Foxp3+ Treg cells. Taken together, these results indicate that administering CVPS as an adjuvant enhances both cellular and humoral immune responses via the TLR-2 and TLR-4 signalling pathways, thereby promoting DC maturation and suppressing TGF-β expression and Treg frequency.

IL-10: master switch from tumor-promoting inflammation to antitumor immunity.

Human cancer is characterized by deficits in antigen-specific immunity and intratumoral CD8(+) T cells. On the other hand, inflammatory macrophages and mediators of chronic inflammation are highly prevalent in patients with late-stage cancer. Intratumoral T-cell deficiency and chronic inflammation have been linked independently to a poor prognosis in patients with cancer, and therapeutic approaches to overcome either pathology separately are in clinical testing. The anti-inflammatory cytokine interleukin (IL)-10 suppresses macrophage and proinflammatory Th17 T-cell responses by inhibiting the inflammatory cytokines IL-6 and IL-12/23. Corroborating the anti-inflammatory action of IL-10, deficiency in IL-10 leads to a stimulation of inflammatory responses and inflammatory bowel disease. The anti-inflammatory role of IL-10 fostered the assumption that IL-10 undermines the immune response to cancer. However, mice and humans deficient in IL-10 signaling develop tumors spontaneously and at high rates. Overexpression of IL-10 in models of human cancer or treatment with a pegylated IL-10 (PEG-IL-10) led to tumor rejection and long-lasting tumor immunity. IL-10 stimulates cytotoxicity of CD8(+) T cells and the expression of IFN-γ in CD8(+) T cells. IL-10-induced tumor rejections are dependent on the expression of IFN-γ and granzymes in tumor-resident CD8(+) T cells and the upregulation of MHC molecules. These findings reconcile earlier clinical data, which showed that recombinant IL-10 increased IFN-γ and granzymes in the blood of treated individuals. PEG-IL-10 is therefore a unique therapeutic agent, which simultaneously stimulates antitumor immunity and inhibits tumor-associated inflammation.

Interleukin-21 Inhibits Humoral Response to an HIV DNA Vaccine by Enhancing Bcl-6 and Pax-5 Expression

Interleukin-21 (IL-21) is a T-cell-derived cytokine that modulates T-cell, B-cell, and natural killer cell responses. It is not known if it could be used as an adjuvant for HIV DNA vaccination. In our study, we investigated if a DNA construct expressing IL-21 (designated as pVAX-IL-21) as a molecule adjuvant could enhance antigen-specific immune responses to an HIV DNA vaccine (pGX-EnvC). We found that a higher level of antigen-specific cytotoxic responses was induced in BALB/C mice immunized with pGX-EnvC with the pVAX-IL-21 via electroporation. The increased response was associated with higher expression of IFN-γ in CD8⁺ T cells. In contrast, the administration of pVAX-IL-21 inhibited the antibody responses to HIV induced by the pGX-EnvC. The plasma cell inhibitory transcription factors B-cell lymphoma 6 protein (Bcl-6) and Pax-5 were increased in B cells from mice that had been immunized by HIV DNA vaccine plus pVAX-IL-21, suggesting that the expressed IL-21 may inhibit the differentiation from B cells to plasma cells. These results indicate that IL-21 could enhance CD8⁺ T-cell immunity, but inhibit humoral responses during HIV DNA vaccination.

Minocycline modulates antigen-specific CTL activity through inactivation of mononuclear phagocytes in patients with HTLV-I associated neurologic disease

BackgroundThe activation of mononuclear phagocytes (MPs), including monocytes, macrophages and dendritic cells, contributes to central nervous system inflammation in various neurological diseases. In HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP), MPs are reservoirs of HTLV-I, and induce proinflammatory cytokines and excess T cell responses. The virus-infected or activated MPs may play a role in immuneregulation and disease progression in patients with HTLV-I-associated neurological diseases.ResultsPhenotypic analysis of CD14+ monocytes in HAM/TSP patients demonstrated high expression of CX3CR1 and HLA-DR in CD14lowCD16+ monocytes, compared to healthy normal donors (NDs) and asymptomatic carriers (ACs), and the production of TNF-α and IL-1β in cultured CD14+ cells of HAM/TSP patients. CD14+ cells of HAM/TSP patients also showed acceleration of HTLV-I Tax expression in CD4+ T cells. Minocycline, an inhibitor of activated MPs, decreased TNF-α expression in CD14+ cells and IL-1β release in PBMCs of HAM/TSP patients. Minocycline significantly inhibited spontaneous lymphoproliferation and degranulation/IFN-γ expression in CD8+ T cells of HAM/TSP patients. Treatment of minocycline also inhibited IFN-γ expression in CD8+ T cells of HAM/TSP patients after Tax11-19 stimulation and downregulated MHC class I expression in CD14+ cells.ConclusionThese results demonstrate that minocycline directly inhibits the activated MPs and that the downregulation of MP function can modulate CD8+ T cells function in HAM/TSP patients. It is suggested that activated MPs may be a therapeutic target for clinical intervention in HAM/TSP.

IL-4 acts as a potent stimulator of IFN-γ expression in CD8+ T cells through STAT6-dependent and independent induction of Eomesodermin and T-bet

CD8+ T cell synthesis of IFN-γ is an important component of the CD8+ T cell immune response. In short-term cultures of murine pan-T cells, we found that IL-4 was the principal cytokine responsible for driving IFN-γ synthesis by CD3/CD28-activated CD8+ T cells. IL-4 was able to induce low levels of IFN-γ mRNA in CD8+ T cells even in the absence of CD3/CD28 engagement, although concomitant CD3/CD28 stimulation was necessary for IFN-γ secretion. IL-4 induction of IFN-γ was explained by its ability to induce Eomesodermin and T-bet transcription factors whose expression was further increased by CD3/CD28. Expression of Eomesodermin, T-bet and IFN-γ induced by IL-4 was partially dependent upon activation of MAPK and PI3K but independent of the canonical IL-4-activated transcription factor, STAT6. In contrast, expression of IFN-γ induced by IL-4/CD3/CD28 stimulation showed additional dependency upon STAT6 which functions to increase expression of Eomesodermin specifically. These novel findings point to a function for IL-4 as a direct regulator of IFN-γ expression in CD8+ T cells and reveal the molecular mechanisms involved.

Alterations of CD4+CD25+Foxp3+ regulatory T cells in HIV‐infected slow progressors of former blood donors in China

Our objective was to study the alterations of CD4(+) CD25(+) Foxp3(+) T(regs) in HIV-infected SPs and to examine the role of T(regs) in the disease progression of HIV. The proportion of CD4(+) CD25(+) Foxp3(+) T(regs) in peripheral blood of 24 SPs, 30 asymptomatic HIV-infected patients, 20 AIDS patients, and 16 non-infected controls was quantified using flow cytometry. HIV Gag peptide mix-induced IFN-γ expression in CD8(+) T cells in whole and CD25-depleted PBMCs was examined to evaluate the function of T(regs) . The expression of CTLA-4 in T(regs) was also detected to measure the suppressive effect of T(regs) . HLA-DR and CD38 expression were measured to study the relationship between the frequency of T(regs) and immune activation of HIV-infected patients. The frequency of CD4(+) CD25(+) Foxp3(+) regulatory T cells in SPs was lower than in asymptomatic HIV-infected patients, AIDS patients, and normal controls (P < 0.05). T(regs) in SPs showed lower intracellular CTLA-4 expression than those of asymptomatic HIV-infected patients and AIDS patients (P < 0.05). The frequency of T(regs) significantly correlated with the percentage of CD38 expression on CD4(+) and CD8(+) T cells (P < 0.05). Multivariate regression analysis showed that the CD4(+) T cell count was the strongest independent factor correlated with the absolute count of T(regs) , while viral load had the strongest predictive strength on the proportion of T(regs) . We conclude that a lower frequency of T(regs) and intracellular CTLA-4 expression of T(regs) was one of the characteristics of SPs that may have important clinical impacts for the prediction of the clinical progress of HIV infection.

PyNTTTTGT Prototype Oligonucleotide IMT504, a Novel Effective Adjuvant of the FMDV DNA Vaccine

Synthetic oligodeoxynucleotides (ODNs) such as CpG can stimulate B and plasmacytoid dendritic cells in vertebrate immune systems. Several studies showed that non-CpG ODNs could also induce strong stimulation of B and T cells. PyNTTTTGT ODNs, non-CpG ODNs, can activate and cause immunoglobulin secretion by B cells and proliferation of T cells in vivo. By using PyNTTTTGT ODNs as an adjuvant for a FMDV DNA vaccine, we found that levels of antibody production, T-cell proliferation, and CTL activity were significantly increased compared with the DNA vaccine alone. Compared with the adjuvant effects of CpG ODNs on DNA vaccination, similar levels of antibody production and T-cell proliferation, and higher levels of CTL activity and IFN-gamma expression in CD8 T cells were induced by the IMT504 ODNs. On the other hand, RT-PCR results show that IMT504 ODN may activate the DNA sensor of DAI (DNA-dependent activator of IFN regulatory factors) and partially stimulate TLR9. At this point, the PyNTTTTGT prototype IMT504 ODN can reasonably be predicted to be a good adjuvant for FMDV DNA vaccine in small animals, but its efficacy in larger animals remains to be explored.

IFN-γ expression in CD8+ T cells regulated by IL-6 signal is involved in superantigen-mediated CD4+ T cell death

Infection with pathogens containing superantigens (Sags) can result in massive excessive CD4+ T cell activation and death in such conditions as toxic shock, food poisoning and autoimmune diseases. We here showed how enhancement of IL-6 signaling suppresses Sag-mediated activated CD4+ T cell death. Sag-induced CD4+ T cell death increased in IL-6 knockout (KO) mice, whereas it decreased in mice characterized by enhanced IL-6-gp130-STAT3 signaling. The serum concentration of IFN-gamma was inversely correlated with the magnitude of IL-6 signaling, and IFN-gamma deficiency inhibited Sag-induced activated CD4+ T cell death, suggesting that IL-6 suppresses CD4+ T cell death via IFN-gamma expression. Interestingly, depletion of activated CD8+ T cells inhibited Sag-mediated increases in IFN-gamma expression in IL-6 KO mice as well as the augmented CD4+ T cell death. The results demonstrate that IL-6-gp130-STAT3 signaling in activated CD8+ T cells contributes to Sag-induced CD4+ T cell death via IFN-gamma expression, highlighting this signaling axis in CD8+ T cells as a potential therapeutic target for Sag-related syndromes.