Increased Production Of Interferon-gamma Research Articles

Abstract CT079: The Pseudovax trial: A novel peptide vaccine targeting mutated GNAS for pseudomyxoma peritonei

Abstract Background: Pseudomyxoma peritonei (PMP) is a rare cancer originating from appendiceal mucinous tumors, which may spread to encompass the entire peritoneal cavity. Complete surgical removal and hyperthermic intraperitoneal chemotherapy cures approximately half of the patients, but patients with recurrent or non-resectable disease have no efficacious treatment options. These patients have a high unmet need for novel treatment options, and in the Pseudovax trial we will explore a novel therapeutic concept. A main molecular driver of PMP (in up to 90% of cases) is otherwise rare mutations in the GNAS oncogene, coding for the Gsα protein. We hypothesized that mutated Gsα would be a tumor neoantigen, and in T cells isolated from peripheral blood of PMP patients, a strong T cell proliferative response was observed. In the corresponding tumors, T cells with up-regulated immune checkpoint molecules PD-1 and TIGIT were detected. Study hypotheses: Patients with GNAS-mutated PMP has a preexisting, attenuated immune response directed against mutated Gsα protein. Vaccination with mutated Gsα peptides can be combined with immune checkpoint inhibition (ICI) with acceptable toxicity. The vaccine will reactivate the immune response and ICI will restore measurable anticancer immunity in patients. Study design: The Pseudovax trial is a small (10 patients) first-in-human, proof-of-concept, prospective, open label, phase I trial. Patients with recurrent or non-resectable PMP are eligible for inclusion. During a treatment period of 12 months, a mutated Gsα peptide vaccine + adjuvant (GM-CSF) will be administered as intradermal injections. After 12 weeks, treatment with ICI will be initiated. The primary endpoints are toxicity and circulating immune response against the vaccine, measured as increase in interferon-gamma production or proliferation of circulating vaccine-specific T cells compared to pre-vaccination baseline. Secondary endpoints are progression-free survival (abdominopelvic CT scans, blood levels of carcinoembryonic antigen (CEA) and/or detection of GNAS mutation by ctDNA analysis). Citation Format: Kjersti Flatmark, Annette Torgunrud, Fleten G. Karianne, Ben Davidson, Hedvig V. Juel, Nadia Mensali, Christin Lund Andersen, Else Marit Inderberg. The Pseudovax trial: A novel peptide vaccine targeting mutated GNAS for pseudomyxoma peritonei [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT079.

Abstract A173: ASP2074, a novel tetraspanin 8 x CD3 bispecific antibody, demonstrates selectivity and antitumor activity in preclinical cancer models

Abstract ASP2074 is a novel bispecific antibody that targets the tumor-associated antigen tetraspanin 8 (TSPAN8) and CD3, which is expressed on the plasma membrane of all T cells. By engaging with both TSPAN8 and CD3, ASP2074 enables the retargeting of cytotoxic T cells specifically against TSPAN8-expressing tumor cells in a human leukocyte antigen-independent manner. TSPAN8 is a member of the tetraspanin superfamily of proteins that plays a crucial role in intracellular signal transduction and regulates various cellular functions, including leukocyte trafficking, angiogenesis, and wound repair. TSPAN8 is highly expressed in various types of cancer, such as colorectal cancer, gastric cancer, pancreatic cancer, esophageal adenocarcinoma, and gastroesophageal junction (GEJ) adenocarcinoma. Its overexpression has been associated with tumor cell growth, angiogenesis, invasion, metastasis, and poor prognosis, making it an attractive target for antibody-based cancer immunotherapy. ASP2074 is currently undergoing phase 1 clinical trials for the treatment of patients with colorectal cancer, esophageal adenocarcinoma, pancreatic cancer, gastric cancer, and GEJ adenocarcinoma. In this study, we examined the selectivity and antitumor activity of ASP2074 in vitro and in vivo. Methods: Retrogenix Cell Microarray Technology was used to evaluate the selective binding of ASP2074 to TSPAN8 and CD3 in 6018 full-length human plasma membrane proteins, secreted and cell surface-tethered human secreted proteins, and 397 human heterodimers containing homologs and isoforms of tetraspanin. Cytotoxicity and T-cell activation of ASP2074 were examined using TSPAN8-expressing tumor cells in combination with human peripheral blood mononuclear cells (PBMCs) as effector cells in a redirected T-cell cytotoxicity assay. Furthermore, the antitumor effect of ASP2074 was examined in a human PBMC-engrafted NOG mouse model bearing human TSPAN8-expressing colorectal cancer cells. Results: Our results showed that ASP2074 selectively bound to TSPAN8 and CD3 without significant binding to other human plasma membrane proteins, secreted proteins, and heterodimers. ASP2074 exhibited cytotoxicity against TSPAN8-expressing cancer cells and increased interferon-gamma production and expression of T-cell activation markers. The cytotoxic activities were dependent on the TSPAN8 expression levels in tumor cells. In the NOG mouse model, ASP2074 exhibited an antitumor effect on human TSPAN8-expressing colorectal cancer cells in vivo. Conclusion: Our findings suggest that ASP2074 has potent antitumor activity through cytotoxicity against TSPAN8-expressing tumor cells by T-cell activation via selective binding to TSPAN8 and CD3. These preclinical results support the continued development of ASP2074 as a promising immunotherapy for the treatment of TSPAN8-expressing cancers. Citation Format: Yoshiyuki Tenda, Yuichi Hanada, Hiroaki Tanaka, Masashi Shimazaki, Kenna Shirasuna. ASP2074, a novel tetraspanin 8 x CD3 bispecific antibody, demonstrates selectivity and antitumor activity in preclinical cancer models [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A173.

Abstract 3197: Adoptive T cell therapy using IL-7 and IL-15 expanded HER2-specific CD4 T cells for metastatic breast cancer

Abstract BACKGROUND: Adoptive cell therapy (ACT) has had limited success due to low T cell infiltration in breast cancer (BC) patients. We have shown that administration of class II HER2 peptide pulsed type I polarized dendritic cell (DC1) vaccines leads to increase in CD4 T cells in the periphery of BC patients. In this study, we investigated the feasibility of expanding CD4 T cells from peripheral blood mononuclear cells (PBMC) of HER2-DC1 vaccinated BC patients. METHODS: PBMCs from BC patients receiving HER2-DC1 vaccines were used for CD4 T cell expansion. PBMCs were co-cultured with HER2-DC1 at 10:1 ratio for initial activation followed by expansion with cytokines- IL-2 and IL-7 or IL-15. Expanded T cells were assessed for immune marker phenotyping by flow cytometry and TCRvb analysis. For TCRvB analysis, pre, post DC1, IL-7 and IL-15 expanded CD4 T cells were pelleted for DNA isolation and sequenced using Immunoseq Analyzer platform. In addition, we investigated the in vivo efficacy of adoptively transferred mouse IL-7/IL-15 expanded Her2 specific CD4 T cells in HER2+ TUBO mouse model. RESULTS: Co-culturing of patient PBMCs with HER2-DC1 in the presence of cytokines IL-2, IL-7 and IL-15 expanded CD4 T cells ranging from 8-43 fold expansion in different samples with 92-98% CD4 phenotype. CD4 T cells expanded in IL-7 had stem like memory phenotype while IL-15 skewed to terminally differentiated CD4 T cells. IL-7 expanded T cells had significantly higher Tim3 and Ox40 expression, and effector memory proportion as compared to the IL-15 expanded T cells. Restimulation of expanded T cells with HER2 pulsed DCs showed HER2 specificity as measured by increased interferon-gamma production We identified top five clones in PBMCs from DC1-HER2 vaccinated BC patients compared to baseline. We observed differential abundance of TCR clones under IL-7 and IL-15 conditions and identified unique clones for IL-7 and IL-15. ACT using a combination of IL-7 and IL-15 expanded mouse CD4 T cells led to 50% tumor regression in HER2+ model. CONCLUSION: These studies demonstrate that HER2 specific CD4 T cells can be successfully expanded from HER2-DC1 vaccinated patients. Post HER2 DC1 vaccine can generate a pool of antigen specific CD4 T cells and may offer a promising ACT for HER2 BC. Further studies are warranted to demonstrate the efficacy in the clinical setting. Citation Format: Namrata Gautam, Krithika Kodumudi, Colin Snyder, Amber Beyer, Ricardo Costa, Heather Han, Brian Czerniecki. Adoptive T cell therapy using IL-7 and IL-15 expanded HER2-specific CD4 T cells for metastatic breast cancer [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 3197.

Abstract 2962: ASP2138, a novel 2+1 format, claudin 18.2 x CD3 bispecific antibody, demonstrates selectivity and activity in preclinical cancer models

Abstract Claudin 18.2 (CLDN18.2) is a member of the claudin family of proteins that are involved in the formation of tight junctions in epithelia and endothelia. In normal tissue, CLDN18.2 is expressed exclusively on gastric epithelial cells. CLDN18.2 expression is maintained during malignant transformation of gastric epithelia. In addition, it is aberrantly expressed in other tumor types, including esophageal, pancreatic, and lung adenocarcinomas. Thus, CLDN18.2 is considered to be an attractive tumor target antigen for antibody-based cancer immunotherapy. ASP2138 is an immunoglobulin G (IgG)-based, asymmetric 2+1 format, T-cell bispecific antibody comprising bivalent humanized anti-CLDN18.2 antigen-binding fragments and a monovalent anti-CD3 single-chain variable fragment. CD3 is a surface protein complex expressed on all T-cell populations and its clustering causes T-cell activation. Therefore, CD3 bispecific antibodies allow for simultaneous engagement with both CD3 and a tumor-associated antigen (TAA), resulting in retargeting of cytotoxic T cells specifically against TAA-positive tumor cells in a human leukocyte antigen-independent manner. ASP2138 is in development for the treatment of patients with CLDN18.2-positive gastric or gastroesophageal junction adenocarcinoma or pancreatic adenocarcinoma, and is being evaluated in an ongoing phase 1/1b clinical trial (NCT05365581). In this study, ASP2138 demonstrated selective binding to CLDN18.2 and CD3 without binding to other homologs and isoforms of CLDN18.2. Cytotoxicity and T-cell activation of ASP2138 were assessed using CLDN18.2-expressing tumor cells in combination with human peripheral blood mononuclear cells (PBMCs) as effector cells in a redirected T-cell cytotoxicity assay in vitro. ASP2138 showed cytotoxicity against CLDN18.2-expressing gastric or pancreatic cancer cells and increased interferon-gamma production and expression of T-cell activation markers. The cytotoxic activities were dependent on the effector cell to target cell ratio and CLDN18.2 expression on tumor cells. ASP2138 exhibited an antitumor effect on human CLDN18.2-expressing gastric cancer in a human PBMC-engrafted NOG mouse model in vivo. In summary, ASP2138 is expected to show a clinical effect through cytotoxicity against CLDN18.2-expressing tumor cells by T-cell activation that results from selective binding to CLDN18.2 and CD3. Citation Format: Taisuke Nakazawa, Hiroaki Tanaka, Aya Kikuchi, Rumana Rashid, Kendra N. Avery, Jing Qi, Alex Nisthal, Masashi Shimazaki, Kenna Shirasuna. ASP2138, a novel 2+1 format, claudin 18.2 x CD3 bispecific antibody, demonstrates selectivity and activity in preclinical cancer 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 2962.

Gas Plasma Technology Augments Ovalbumin Immunogenicity and OT-II T Cell Activation Conferring Tumor Protection in Mice.

Reactive oxygen species (ROS/RNS) are produced during inflammation and elicit protein modifications, but the immunological consequences are largely unknown. Gas plasma technology capable of generating an unmatched variety of ROS/RNS is deployed to mimic inflammation and study the significance of ROS/RNS modifications using the model protein chicken ovalbumin (Ova vs oxOva). Dynamic light scattering and circular dichroism spectroscopy reveal structural modifications in oxOva compared to Ova. T cells from Ova‐specific OT‐II but not from C57BL/6 or SKH‐1 wild type mice presents enhanced activation after Ova addition. OxOva exacerbates this activation when administered ex vivo or in vivo, along with an increased interferon‐gamma production, a known anti‐melanoma agent. OxOva vaccination of wild type mice followed by inoculation of syngeneic B16F10 Ova‐expressing melanoma cells shows enhanced T cell number and activation, decreased tumor burden, and elevated numbers of antigen‐presenting cells when compared to their Ova‐vaccinated counterparts. Analysis of oxOva using mass spectrometry identifies three hot spots regions rich in oxidative modifications that are associated with the increased T cell activation. Using Ova as a model protein, the findings suggest an immunomodulating role of multi‐ROS/RNS modifications that may spur novel research lines in inflammation research and for vaccination strategies in oncology.

Conversion of ATP to adenosine by CD39 and CD73 in multiple myeloma can be successfully targeted together with adenosine receptor A2A blockade

BackgroundPD1/PDL1-directed therapies have been unsuccessful for multiple myeloma (MM), an incurable cancer of plasma cells in the bone marrow (BM). Therefore, other immune checkpoints such as extracellular adenosine and its...

T-cells dysregulate hematopoiesis in Myeloproliferative Neoplasm through increased interferon-gamma production

Abstract Chronic inflammation is a key feature of myeloproliferative neoplasm (MPN), a hematological malignancy. MPN is characterized acquisition of a somatic mutation in hematopoietic stem cells, most commonly the JAK2V617F mutation, which leads to unrestrained expansion of myeloid cells. Although lymphoid compartments do not have the JAK2V617F mutation, these cells are key mediators of inflammation. The precise role of lymphoid cells in disease pathogenesis is unclear. We found that CD4+ T cells from MPN patients produce significantly more interferon gamma (IFNg) than normal CD4+ T cells. IFNg has been reported to suppress the growth of myeloid colonies. We found that removal of T cells from peripheral blood mononuclear cells of MPN patients enhance myeloid colony formation. Removal of T cell also increase fraction of wild-type myeloid colonies, demonstrating that wild-type cells are more sensitive to suppression by T cells. In addition, MPN mouse model using transduction-transplantation of JAK2V617F in RAG2−/− mice, lacking in T and B cells, display an attenuated MPN phenotype. Moreover, transplantation of JAK2V617F RAG2−/− bone marrow with wild-type bone marrow into lethally irradiated wild-type recipient restored the MPN pathology, such as erythrocytosis and expansion of megakaryocytes. This implicates that lymphocytes play an important role in driving MPN pathogenesis. More importantly, inhibition of T cells in JAK2V617F transplanted mouse using cyclosporine attenuates spleen size and marrow fibrosis, demonstrating that suppression of T cells after disease has already been established is therapeutic. In sum, these data shows that T mediates pathogenesis in MPN, and that suppressing T cells in MPN may have therapeutic benefit.

Modulation of T-Cell Function in the Microenvironment of Emu-TCL1 CLL Bearing Mice By Btki Appears Independent of ITK

Background: Chronic lymphocytic leukemia (CLL) development is associated with global immunodeficiency including T-cell exhaustion. We hypothesise that repairing T cell functions would improve outcome and decrease infectious complications which cause significant morbidity in CLL patients. Chronic B-cell receptor (BCR) activation as well as close interactions with the tumour microenvironment promote survival of malignant CLL B-cells, supporting their ability to induce immune suppression. To date, the most clinically successful approach to BCR-signalling inhibition is by the use of BTK inhibitors (BTKi). It has been suggested that the BTKi Ibrutinib has the ability to modulate T-helper cell polarity from Th2 to Th1 and thus would be a step towards repairing CLL associated T-cell defects (1). We were therefore interested to determine whether the second generation BTKi Acalabrutinib which has no reported inhibitory capacity towards ITK would have similar effects than Ibrutinib in modulating T cell responses.Materials and Methods: To address this question in vivo C57/Bl6 animals 2.5 months of age were injected with 40x10e6 purified CLL B-cells pooled from Eµ-TCL1 mice with CLL. When peripheral blood CLL load reached >10% animals were randomized (mean day 14) to either vehicle treatment (2% HPBD) or Acalabrutinib treatment (0.15 mg/l in 2% HPBC) for 21 days. 17 animals were group were analysed. Splenic cells were isolated, the cellular component characterized by CyTOF and T cell function assessed by multi-parameter flow cytometry and T-cell synapse formation assay.Results: Treatment with Acalabrutinib resulted in increased expression of IL2 (p<0.0001) in CD4+ T cells and decreased expression of IL4 among both CD4+ T cells (p=0.0016) but not CD8+ T-cells. There was a reduction in Interferon gamma production in both CD4 T-cells (p=0.0463) and CD8+ T-cells (p=0.0064) with Acalabrutinib treatment. In addition, treatment resulted in an increase in CD107a+/CD107a- ratio among both CD44+ and CD44- CD8+ cytotoxic T-cells. This effect was pronounced in the antigen experienced CD44+ cytotoxic T-cells (p<0.0001) but only moderate (p=0.0056) in the CD44- cytotoxic T-cells. Lastly, we find a statistically significant increase in T-cell synapse area (p<0.0001) with Acalabrutinib treatment (Figure 1).Conclusion: We find that treatment with both Ibrutinib and Acalabrutinib result in a similar shift of T cell function with cytokine secretion with increased IL2 and decreased IL4. T-cells in CLL have increased Interferon gamma production (2) and the observed decrease seen with Acalabrutinib is in keeping with a normalization of T cell function. Moreover, overall CD8+ T-cell function is increased with Acalabrutinib treatment as evidenced by an increase in cytotoxic T-cell function and immune synapse formation. We speculate that inhibition of ITK is not the leading cause for this phenomenon as Acalabrutinib does not have inhibitory capacity toward this kinase. These changes suggest that BTKi modulate T cell mediated immune responses indirectly via either their effects in the CLL B-cell or myeloid cells in the tumour microenvironment.ReferencesDubovsky JA, Beckwith KA, Natarajan G, Woyach JA, Jaglowski S, Zhong Y, et al. Ibrutinib is an irreversible molecular inhibitor of ITK driving a Th1-selective pressure in T lymphocytes. Blood. 2013;122(15):2539-49.Riches JC, Davies JK, McClanahan F, Fatah R, Iqbal S, Agrawal S, et al. T cells from CLL patients exhibit features of T-cell exhaustion but retain capacity for cytokine production. Blood. 2013;121(9):1612-21. [Display omitted] DisclosuresFrigault:Acerta Pharma: Employment. Gribben:Abbvie: Honoraria; Acerta Pharma: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Wellcome Trust: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Unum: Equity Ownership; Roche: Honoraria; TG Therapeutics: Honoraria; NIH: Research Funding; Medical Research Council: Research Funding; Cancer Research UK: Research Funding; Kite: Honoraria; Pharmacyclics: Honoraria; Novartis: Honoraria.

Synergistic Antitumor Effect on Bladder Cancer by Rational Combination of Programmed Cell Death 1 Blockade and CRISPR-Cas9-Mediated Long Non-Coding RNA Urothelial Carcinoma Associated 1 Knockout.

Targeted therapy produces objective responses in bladder cancer patients, although the responses can be short. Meanwhile, response rates to immune therapy are lower, but the effects are more durable. Based on these findings, it was hypothesized that urothelial carcinoma associated 1 (UCA1)-targeted therapy could synergize with programmed cell death 1 (PD-1) blockade to enhance antitumor activity. To test this hypothesis, the effects of CRISPR-Cas9 targeting of UCA1 and PD-1 were assessed in vitro and in vivo. It was found that gRNA/cas9-targeted UCA1 induced apoptosis of 5637 bladder cancer cells, whereas PD-1 gene knockout could be achieved by electroporation of gRNA/cas9 targeting PD-1, as detected by polymerase chain reaction. In 5637 cell-xenografted humanized SCID mice, stimulation with CRISPR-Cas9 systems, immune phenotypes, and cytokine expression of human dendritic cells (DCs) was detected by flow cytometry, and polymerase chain reaction, respectively. The results of these assays suggested that the gRNA/cas9 treatment upregulated expression of CD80, CD83, and CD86 and significantly increased interleukin (IL)-6, IL-12, and IL-23 and tumor necrosis factor alpha mRNA levels. Co-administration of anti-PD-1 and anti-UCA1 treatment suppressed tumor growth and markedly improved survival of 5637 xenografted mice. Additionally, the combination treatment increased interferon gamma production by T cells that subsequently enhanced the expression of Th1-associated immune-stimulating genes to reduce transcription of regulatory/suppressive immune genes and reshape the tumor microenvironment from an immunosuppressive to a stimulatory state. Finally, anti-UCA1 treatment was shown to induce interferon gamma-dependent programmed cell death ligand 1 expression within 5637 xenograft tumors in vivo. Together, these results demonstrate potent synergistic effects of a combination therapy using LncRNA UCA1-targeted therapy and immune checkpoint blockade of PD-1, thus supporting the translational potential of this combination strategy for clinical treatment of bladder cancer.

The Four-Way Stop Sign: Viruses, 12-Lipoxygenase, Islets, and Natural Killer Cells in Type 1 Diabetes Progression.

Natural killer (NK) cells represent an important effector arm against viral infection, and mounting evidence suggests that viral infection plays a role in the development of type 1 diabetes (T1D) in at least a portion of patients. NK cells recognize their target cells through a delicate balance of inhibitory and stimulatory receptors on their surface. If unbalanced, NK cells have great potential to wreak havoc in the pancreas due to the beta cell expression of the as-yet-defined NKp46 ligand through interactions with the activating NKp46 receptor found on the surface of most NK cells. Blocking interactions between NKp46 and its ligand protects mice from STZ-induced diabetes, but differential expression non-diabetic and diabetic donor samples have not been tested. Additional studies have shown that peripheral blood NK cells from human T1D patients have altered phenotypes that reduce the lytic and functional ability of the NK cells. Investigations of humanT1D pancreas tissues have indicated that the presence of NK cells may be beneficial despite their infrequent detection. In non-obese diabetic (NOD) mice, we have noted that NK cells express high levels of the proinflammatory mediator 12/15-lipoxygenase (12/15-LO), and decreased levels of stimulatory receptors. Conversely, NK cells of 12/15-LO deficient NOD mice, which are protected from diabetes development, express significantly higher levels of stimulatory receptors. Furthermore, the human NK92 cell line expresses the ALOX12 protein [human 12-lipoxygenase (12-LO), related to mouse 12/15-LO] via Western blotting. Human 12-LO is upregulated in the pancreas of both T1D and T2D human donors with insulin-containing islets, showing a link between 12-LO expression and diabetes progression. Therefore, our hypothesis is that NK cells in those susceptible to developing T1D are unable to function properly during viral infections of pancreatic beta cells due to increased 12-LO expression and activation, which contributes to increased interferon-gamma production and an imbalance in activating and inhibitory NK cell receptors, and may contribute to downstream autoimmune T cell responses. The work presented here outlines evidence from our lab, as well as published literature, supporting our hypothesis, including novel data.

Leukocyte Bim deficiency does not impact atherogenesis in ldlr\u2212/\u2212 mice, despite a pronounced induction of autoimmune inflammation

Proapoptotic Bcl-2 family member Bim is particularly relevant for deletion of autoreactive and activated T and B cells, implicating Bim in autoimmunity. As atherosclerosis is a chronic inflammatory process with features of autoimmune disease, we investigated the impact of hematopoietic Bim deficiency on plaque formation and parameters of plaque stability. Bim−/− or wild type bone marrow transplanted ldlr−/− mice were fed a Western type diet (WTD) for 5 or 10 weeks, after which they were immunophenotyped and atherosclerotic lesions were analyzed. Bim−/− transplanted mice displayed splenomegaly and overt lymphocytosis. CD4+ and CD8+ T cells were more activated (increased CD69 and CD71 expression, increased interferon gamma production). B cells were elevated by 147%, with a shift towards the pro-atherogenic IgG-producing B2 cell phenotype, resulting in a doubling of anti-oxLDL IgG1 antibody titers in serum of bim−/− mice. Bim−/− mice displayed massive intraplaque accumulation of Ig complexes and of lesional T cells, although this did not translate in changes in plaque size or stability features (apoptotic cell and macrophage content). The surprising lack in plaque phenotype despite the profound pro-atherogenic immune effects may be attributable to the sharp reduction of serum cholesterol levels in WTD fed bim−/− mice.

Association of Ribonuclease T2 Gene Polymorphisms With Decreased Expression and Clinical Characteristics of Severity in Crohn’s Disease

Variants in the tumor necrosis factor superfamily member 15 gene (TNFSF15, also called TL1A) have been associated with risk for inflammatory bowel disease (IBD). TL1A affects expression of multiple cytokines to promote mucosal inflammation. Little is known about the TL1A-response pathways that regulate cytokine expression. We investigated T-cell gene expression patterns to determine the mechanisms by which TL1A regulates cytokine production, and whether these associate with outcomes of patients with Crohn's disease (CD). Peripheral T cells isolated from normal donors were cultured with TL1A. We performed gene expression profile analysis by RNA sequencing of subsets of interferon gamma (IFNG)-producing and non-producing cells purified by flow cytometry. Unsupervised hierarchical clustering analysis was used to identify gene expression differences between these subsets. Ribonuclease T2 gene (RNASET2) expression and methylation were assessed by quantitative trait loci analyses. Clinical characteristics of patients (complications, resistance to therapy, and recurrence time) were associated with single nucleotide polymorphisms in RNASET2. We performed motif screening to identify polymorphisms that disrupt transcription factor binding sites. Levels of RNASET2 were knocked down with small interfering RNA in CD4+ T cells and the effect on protein expression was determined by proteomic analysis and cytokine production. Cell aggregation was measured by flow cytometry. We identified 764 genes with at least a 2-fold difference in TL1A-mediated expression between IFNG-secreting and non-secreting T cells (P < 1× 10-5). Many of these genes were located near IBD susceptibility variants. RNASET2 was the only IBD risk-associated gene with >5-fold down-regulation in the IFNG-secreting subset. RNASET2 disease risk variants were associated with decreased expression in peripheral and mucosal tissues and DNA hypermethylation in CD patients requiring surgical intervention. RNASET2 disease risk variants were associated in CD patients with more complicated disease or resistance to therapy, defined in part by failed response to treatment, increased length of intestinal resection, shorter time to repeat surgery, and high Rutgeerts score (>2) in postoperative endoscopy. The RNASET2 variant rs2149092 was predicted to disrupt a consensus binding site for the transcription factor ETS within an enhancer region. Expression of RNASET2 correlated with expression of ETS. RNASET2 knockdown in T cells increased expression of IFNG and intercellular adhesion molecule 1 (ICAM1) and induced T-cell aggregation. A blocking antibody against (ILFA1), disrupting the lymphocyte function-associated antigen 1-intercellular adhesion molecule 1 interaction, reduced T-cell production of IFNG. We identified decreased expression of RNASET2 as a component of TL1A-mediated increase in production of IFNG and as a potential biomarker for patients with severe CD. Further study of the role of RNASET2 in regulating mucosal inflammation may lead to development of novel therapeutic targets.

Epitope analysis and protection by a ROP19 DNA vaccine againstToxoplasma gondii

We used bioinformatics approaches to identify B-cell and T-cell epitopes on the ROP19 protein of Toxoplasma gondii. Then, we constructed plasmids with ROP19 (pEGFP-C1-ROP19) and injected them into BALB/c mice to test the immunoprotection induced by this vaccine candidate. The results showed that immunization with pEGFP-C1-ROP19 induced effective cellular and humoral immune responses in mice; specifically, high serum levels of T. gondii-specific IgG and increased interferon-gamma production by splenocytes. Furthermore, the mice vaccinated with pROP19 had significantly fewer brain cysts (583 ± 160) than the mice injected with phosphate-buffered saline (1350 ± 243) or with the control plasmid, pEGFP-C1 (1300 ± 167). Compared with PBS-treated mice, those immunized with pROP19 had only 43% of the number of brain cysts. These results suggest that the DNA vaccine encoding ROP19 induced a significant immune response and provided protection against a challenge with T. gondii strain PRU cysts.

Activation of Natural Killer Cells During Acute Infection With Hepatitis C Virus

Natural killer (NK) cells are essential early after infection, not only for viral containment but also for timely and efficient induction of adaptive responses. An inhibitory effect of hepatitis C virus (HCV)-E2 proteins on NK cells has been reported, but the features of NK cell responses in the acute phase of hepatitis C are still largely undefined. Therefore, the aim of this study was to characterize the function and phenotype of NK cells in the acute phase of infection and compare individuals with chronic and self-limited outcomes. Twenty-two individuals with acute HCV infection, 14 with chronic evolution, and 8 with self-limited infection, were studied using NK phenotypic and functional assays. An increased expression of NKG2D on both CD56(bright) and CD56(dim) NK cells was detected in patients with acute HCV, irrespective of the outcome, as compared with healthy controls. Also, interferon gamma production and cytotoxicity by NK cells were higher in individuals with acute HCV infection than in healthy controls. Subset analysis showed increased interferon gamma production in both NK cell subsets carrying group 1 and group 2 HLA-C-specific killer cell immunoglobulin-like receptors. However, increased CD107a was noted only on NK cells expressing the group 1 HLA-C-specific killer cell immunoglobulin-like receptor and was maximal in self-limited infection. Our data show that in the acute phase of HCV infection, NK cells are activated regardless of outcome, with no evidence of a suppressive effect of HCV on NK cell function.

Immunological Response to Renal Cryoablation in an In Vivo Orthotopic Renal Cell Carcinoma Murine Model

The immunological consequences of cryoablation for renal cell carcinoma are largely unknown. Cryoablation is an attractive therapeutic option for tumors due to its minimally invasive nature. Cryoablation is also potentially immunogenic. We describe the development of an animal model to deliver in vivo renal cryotherapy to orthotopically implanted renal cell carcinoma and the results of multiple immunological interrogations after cryoablation. Four to 6-week-old female Balb/c mice (Jackson Laboratories, Bar Harbor, Maine) underwent renal subcapsular implantation of the syngeneic murine renal cell carcinoma Renca. Two weeks later contact cryoablation was done in tumor bearing kidneys. Another group of animals underwent cryoablation of normal kidneys. Animals were sacrificed 2 weeks after tumor injection or 1 and 2 weeks after cryoablation, respectively. Kidneys, spleens and draining lymph nodes were harvested. Evaluation consisted of immunohistochemistry, immunofluorescence and gene expression profiling using reverse-transcriptase polymerase chain reaction. Subcapsular tumor implantation was successful in all cases and confirmed histologically. No significant lymphocytic infiltrate was seen in tumor only animals but those treated with cryoablation (tumor and nontumor bearing) had a significant inflammatory response primarily in sublethal tissue injury and perivascular areas. After cryoablation most infiltrating cells were neutrophils, macrophages and T cells. Polymerase chain reaction showed increased interferon-gamma production in kidneys after cryoablation. This study shows the potential feasibility of this animal model for studying cryo-immunology. We confirm the absence of any significant immune cell infiltration in tumor bearing kidneys and report a significant inflammatory infiltrate after cryoablation, consisting primarily of neutrophils, macrophages, and CD4+ and CD8+ T cells with an increase in the T helper type 1/2 ratio. This orthotopic murine model can form the basis of future studies of additional immunological aspects of renal cryoablation.

A NovelBacillus Calmette-Guérin-based Breast Cancer Vaccine that Coexpresses Multiple Tandem Repeats of MUC1 and CD80 Breaks the Immune Tolerance and Inhibits MUC1-Positive Breast Cancer Growth

Many approaches targeting MUC1 for breast tumor immunotherapy have been attempted. However, preclinical trials with MUC1 showed that MUC1 is a relatively poor immunogen in human. B7 molecules that bind CD28 provide an antigen-nonspecific signal, which, along with an antigen-specific signal, is crucial for T-cell activation. In the present study, we constructed a novel Bacillus Calmette-Guérin-based breast cancer vaccine that coexpressed four VNTRs (variable-number tandem repeats) of MUC1 and CD80 (rBCG-MVNTR4-CD80). The aim of our study was to enhance anti-MUC1 tumor immunity by vaccination of hu-PBL-SCID mice with the recombinant BCG vaccine. The inhibition effect on tumors from the mice immunized with rBCG-MVNTR4-CD80 significantly increased, compared with rBCG-MVNTR4, BCG-pDE22, and phosphate-buffered saline immunized mice (p < 0.05, p < 0.05, p < 0.05). ELISpot assays showed that there was a significant increase in interferon-gamma production in the splenocytes from the mice immunized with rBCG-MVNTR4-CD80. In addition, CD4 and CD8-positive lymphocytes in tumors from rBCG-MVNTR4-CD80-immunized animals were detected. These data showed that rBCG-MVNTR4-CD80 immunization elicited tumor-specific immune response, which closely related with the B7 molecule (CD80), indicating that the vaccine may be a good candidate for MUC1-positive breast cancer immunotherapy.

Single-Chain Antibodies That Target Lentiviral Vectors to MHC Class II on Antigen-Presenting Cells

Lentiviral vectors are promising vaccines because they can transduce and express antigens in dendritic cells in vivo, leading to potent immunization. To improve the safety and efficacy of lentivector vaccination, we sought to target vector transduction to antigen-presenting cells by modifying the viral envelope. To do this we screened a nonimmunized human single-chain antibody phage display library for phage that bound mouse bone marrow-derived dendritic cells (BMDCs) and isolated three single-chain antibodies (scFvs) that bound to more than 20% of cells in the BMDC culture. The three scFvs also bound to dendritic cells, macrophages, monocytes, and B cells from mouse spleen, but not to neutrophils, eosinophils, or T cells. Immunoblotting demonstrated that two unique scFvs, C2 and C7, recognized MHC class II. We constructed chimeric envelope proteins, by fusing these two scFvs to the amino terminus of the amphotropic murine leukemia virus envelope (MLV-A). These chimeric envelopes were expressed on the surface of lentiviral vector particles and enhanced infection (5- to 10-fold) of BMDC cultures, compared with lentiviral vectors with unmodified MLV-A envelope. Similarly, the chimeric envelopes enhanced (10- to 20-fold) the infection of primary lymph node class II-positive cells. One of the envelopes, C2, gave increased interferon-gamma production from splenocytes of vaccinated mice compared with MLV-A, achieving a level similar to that obtained with vesicular stomatitis virus glycoprotein G, when used to deliver an ovalbumin model antigen gene. These results demonstrate that surface-targeting lentiviral vector transduction of antigen-presenting cells gives efficient and potentially safer immunization.

AFCo1, a meningococcal B-derived cochleate adjuvant, strongly enhances antibody and T-cell immunity against Plasmodium falciparum merozoite surface protein 4 and 5

BackgroundWhilst a large number of malaria antigens are being tested as candidate malaria vaccines, a major barrier to the development of an effective vaccine is the lack of a suitable human adjuvant capable of inducing a strong and long lasting immune response. In this study, the ability of AFCo1, a potent T and B cell adjuvant based on cochleate structures derived from meningococcal B outer membrane proteoliposomes (MBOMP), to boost the immune response against two Plasmodium falciparum antigens, merozoite surface protein 4 (MSP4) and 5 (MSP5), was evaluated.MethodsComplete Freund's adjuvant (CFA), which is able to confer protection against malaria in animal MSP4/5 vaccine challenge models, was used as positive control adjuvant. MSP4 and 5-specific IgG, delayed-type hypersensitivity (DTH), T-cell proliferation, and cytokine production were evaluated in parallel in mice immunized three times intramuscularly with MSP4 or MSP5 incorporated into AFCo1, synthetic cochleate structures, CFA or phosphate buffered saline.ResultsAFCo1 significantly enhanced the IgG and T-cell response against MSP4 and MSP5, with a potency equivalent to CFA, with the response being characterized by both IgG1 and IgG2a isotypes, increased interferon gamma production and a strong DTH response, consistent with the ability of AFCo1 to induce Th1-like immune responses.ConclusionGiven the proven safety of MBOMP, which is already in use in a licensed human vaccine, AFCo1 could assist the development of human malaria vaccines that require a potent and safe adjuvant.

Imatinib mesylate, a new kid on the block for the treatment of anti-neutrophil cytoplasmic autoantibodies-associated vasculitis?

Persistent T cell activation is a common finding in anti-neutrophil cytoplasmic autoantibodies (ANCA)-associated systemic vasculitis (AAV) patients. Because imatinib, a selective inhibitor of the ABL, ARG, PDGFR and c-KIT tyrosine kinases, inhibits T cell activation, this study was conducted to evaluate the potential use of imatinib for the treatment AAV patients refractory to conventional therapy. In particular, we investigated the inhibition of T cell activation by this drug and its efficacy on activated T cells from anti-neutrophil cytoplasmic autoantibodies (ANCA)-associated systemic vasculitides (AASV) patients. T cell stimulation has been induced by anti-CD3/anti-CD28 antibodies or by phorbol myristate acetate (PMA)/ionomycin. T cell proliferation was analysed by tritiumthymidine incorporation. Cell cycle progression was determined by propidium iodide staining using fluorescence activated cell sorter (FACS) analysis and by RNAse protection assay (RPA). Cytokine levels were assessed by enzyme-linked immunosorbent assay. T cell proliferation was inhibited significantly by imatinib, due most probably to cell cycle arrest in the G1-phase. This was paralleled by inhibition in the expression of cyclin-dependent kinases 1 and 2 mRNA. The expression of CD25 in naive and memory T cells was decreased significantly by imatinib in activated T cells. Similarly, conversion from naive to memory T cells after T cell activation was impaired by imatinib. Imatinib did not influence interleukin-2 and tumour necrosis factor-alpha production but increased interferon-gamma production. These observed effects of imatinib were similar in T cells from AASV patients and from healthy individuals. Imatinib might be an alternative therapeutical option for AASV patients refractory to conventional therapy.

Structured treatment interruptions (STIs) in HIV-1 infected pediatric populations increases interferon gamma production and reduces viremia

We assessed the effect of progressively longer antiretroviral structured treatment interruptions (STIs) starting with 3 days, increasing by 2 days in length each cycle on HIV-specific immune responses. As well, we correlated these responses with control of HIV viremia. Eight individuals became viremic and reached cycle 13 with an STI of ≥27 days. HIV-specific gamma-interferon production to inactivated HIV and vaccinia vectors expressing gag, env, nef, and pol increased (>10-fold) in six of eight subjects. Median plasma RNA levels peaked @ cycle 7 and declined to levels <10 4 cp/ml after cycle 10. In a subset of five who reached cycle 17, HIV-specific IFN-gamma frequencies increased from cycle 8 to cycle 17 with evidence of improved virologic control over comparable periods off antiretroviral therapy. This allowed us to conclude that exposure to autologous virus increased HIV-specific immune responses and decreased HIV RNA were seen in those who have had >13 interruptions, with STI intervals that exceeded 27 days.