Recombinant Adenovirus Expressing a Soluble Fusion Protein PD-1/CD137L Subverts the Suppression of CD8+ T Cells in HCC

Immunity against hepatitis B virus (HBV) infection is complex and not entirely understood so far, including the decisive factors leading to the development of chronic hepatitis B. This lack of a mechanistic understanding of HBV-specific immunity is also caused by a limited number of suitable animal models. Here, we describe the generation of a recombinant adenovirus expressing an HBV 1.3-overlength genome linked to luciferase (Ad-HBV-Luc) allowing for precise analysis of the quantity of infected hepatocytes. This enables sensitive and close-meshed monitoring of HBV-specific CD8 T cells and the onset of anti-viral immunity in mice. A high dose of Ad-HBV-Luc developed into chronic hepatitis B accompanied by dysfunctional CD8 T cells characterized by high expression of PD1 and TOX and low expression of KLRG1 and GzmB. In contrast, a low dose of Ad-HBV-Luc infection resulted in acute hepatitis with CD8 T cell-mediated elimination of HBV-replicating hepatocytes associated with elevated sALT levels and increased numbers of cytotoxic HBV-specific CD8 T cells. Thus, the infectious dose was a critical factor to induce either acute self-limited or chronic HBV infection in mice. Taken together, the new Ad-HBV-Luc vector will allow for highly sensitive and time-resolved analysis of HBV-specific immune responses during acute and chronic infection.

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Abstract 2577: RLI, a sushi-IL-15Rα/IL-15 fusion protein, is a potent immunomodulatory agent on NK and CD8+ T cells and synergizes with anti-PD1 treatment in preclinical mouse tumor models

10.1158/1538-7445.am2014-2577 ◽

2014 ◽

Author(s):

Mélanie Desbois ◽

Coralie Beal ◽

Clelia Coutzac ◽

Magali Terme ◽

Geraldine Teppaz ◽

...

Keyword(s):

T Cells ◽

Fusion Protein ◽

Cd8 T Cells ◽

Mouse Tumor ◽

Tumor Models ◽

Immunomodulatory Agent ◽

Mouse Tumor Models

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LMP2-Cytotoxic T Lymphocyte Therapy for Relapsed EBV Positive Lymphoma.

Blood ◽

10.1182/blood.v104.11.3276.3276 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3276-3276

Author(s):

Catherine M. Bollard ◽

Helen M. Huls ◽

Karin C. Straathof ◽

Stephen M. Gottschalk ◽

Teresita Lopez ◽

...

Keyword(s):

T Cells ◽

Cd8 T Cells ◽

Stable Disease ◽

Peripheral Blood ◽

Recombinant Adenovirus ◽

Genetically Modified ◽

Fold Increase ◽

Residual Tumor ◽

Cell Transplant ◽

Type Ii

Abstract EBV-associated Hodgkin’s Disease (HD) and non-Hodgkins Lymphoma (NHL) show type II latency. They express the subdominant EBV antigens EBNA1, LMP1 and LMP2, which may serve as targets for immunotherapy approaches. In our previous studies, we used polyclonal EBV-specific CTL (EBV-CTL) in patients with relapsed EBV +ve HD and saw 2 complete and 1 partial response in 11 patients. Tetramer and functional analyses of EBV-CTL lines showed that small populations of T cells reactive against the tumor-associated antigen LMP2 were present in the majority of the infused lines, with some expansion in the peripheral blood following infusion. We therefore hypothesized that CTL specifically targeting type II viral latency antigens might have greater efficacy in these patients. LMP2-CTL could be generated from normal donors using dendritic cells (DC) genetically modified with a recombinant adenovirus encoding LMP2a (Ad5LMP2a) to direct the CTL response to LMP2. However, this approach required the generation of large numbers of DC to expand LMP2-CTL and was not practical in these heavily pretreated patients. We therefore modified the LMP2-CTL generation protocol to use DC for the initial stimulation, followed by stimulation with LCL that had been genetically modified to overexpress LMP2a by transduction with an Ad5f35LMP2a vector. This approach allowed us to specifically expand LMP2-CTL from patients to the numbers required for clinical use. We have generated LMP2 specific CTL lines in 10 patients with EBV+ve HD or NHL. LMP2 peptide tetramers were used for analysis of the polyclonal LMP2-CTL lines where HLA-restricted tetramers were available, and the lines recognized 2–6 (median 4) LMP2 epitopes, as determined by ELISPOT assay. A mean of 22.8% (5–42.1%) of CD8+ T cells bound these LMP2 tetramers, compared to a mean of 0.11% (0.01–0.24%) of LMP2-tetramer positive CD8+ T cells found in CTL generated with genetically unmodified LCL from the same patients. So far, 6 patients have been treated, initially receiving 2 doses of 2x107 CTL/m2 two weeks apart. All patients were off other therapies for at least 1 month prior to receiving CTL. No immediate toxicity was observed. In patients with identified LMP2-epitopes, measurement of IFNγ secretion by CD8+ T cells after stimulation with appropriate LMP2-peptides in ELISPOT assays showed a 4–25-fold increase in spot forming cells after infusions. In contrast, the frequency of CMV and superantigen-specific T cells did not increase. Four patients without radiological evidence of disease who received CTL as adjuvant therapy post stem cell transplant or chemotherapy remain well up to 9 months post CTL. Two patients with measurable disease at the time of CTL infusion had stable disease 8 weeks post CTL. They received 2 further doses of LMP2-CTL at 2x107/m2/dose. Re-evaluation was performed 8 weeks after the additional CTL infusions and one patient continues with stable disease. In the second patient, radiological review now revealed no evidence of disease, and a supraclavicular lymph node resection showed selective accumulation of LMP2-tetramer positive cells (0.3% compared to 0.01% in the peripheral blood) with scanty residual tumor cells. Immunotherapy with autologous LMP2-CTL is therefore well tolerated in patients with relapsed EBV+ve HD/NHL and infused LMP2-CTL cells can localize to the tumor and induce a clinical response.

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ITK-SYK Induces T-Cell Lymphoma with Accompanying Inflammatory Disease in Mice.

Blood ◽

10.1182/blood.v110.11.693.693 ◽

2007 ◽

Vol 110 (11) ◽

pp. 693-693

Author(s):

Christine Dierks ◽

Hong Ma ◽

Hendrik Veelken ◽

Markus Warmuth ◽

Francisco Adrian

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

Fusion Protein ◽

Cd8 T Cells ◽

Point Mutations ◽

Kinase Domain ◽

Pleckstrin Homology Domain ◽

T Cell Lymphomas ◽

Malignant T Cells

Abstract The ITK-SYK fusion protein was previously described to be present in 17% of unspecified peripheral T-cell lymphomas. Here we demonstrate that expression of ITK-SYK in the bone marrow of Balb\c mice causes T-cell lymphomas in mice with a latency of only 3–4 weeks. The disease is characterized by infiltration of the spleen, lymph nodes, bone marrow and the skin with malignant T-cells and progredient destruction of these organs. The mice die about 2 months after the transplantation due to dramatic weight loss caused by infiltration of T-cells into the colon and because of progredient anemia end neutropenia due to progredient infiltration of the bone marrow. The malignant T-cells were characterized as a mixed population of CD3+, CD4+, CD8- T cells and CD3+, CD4-, CD8- T-cells. The malignant disease was accompanied by a generalized inflammatory reaction including upregulation of the inflammatory cytokines IL-5 and INF-γ. Modulation of the membrane binding of ITK-SYK or its binding to Cbl by point mutations in the pleckstrin homology domain of ITK could alter the transforming activity of ITK-SYK. The intact kinase domain was essential for the transformation process and the disease could be reversed by treatment of diseased mice with the Syk-inhibitor Curcumine. Our results demonstrate that the fusion protein ITK-SYK causes T-cell lymphomas in mice and mimics the human disease. Therefore pharmacological inhibition of Syk in patients with U-PTCL carrying the ITK-SYK fusion protein might be a new and effective treatment strategy.

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The Fusion Protein of CTP-HBcAg18-27-Tapasin Mediates the Apoptosis of CD8+T Cells and CD8+ T Cell Response in HLA-A2 Transgenic Mice

Hepatitis Monthly ◽

10.5812/hepatmon.16161 ◽

2014 ◽

Vol 14 (2) ◽

Cited By ~ 4

Author(s):

Yu-Yan Tang ◽

Zheng-Hao Tang ◽

Yi Zhang ◽

Meng Zhuo ◽

Guo-Qing Zang ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Transgenic Mice ◽

Fusion Protein ◽

Cd8 T Cells ◽

Cell Response ◽

Cd8 T Cell ◽

T Cell Response

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Activation of NK and CD8+ T-Cells with a Novel IL-15 and TGF-Beta Receptor Fusion Protein Confers Anti-Tumor Immunity

Blood ◽

10.1182/blood.v126.23.3421.3421 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3421-3421

Author(s):

Spencer Ng ◽

Jiusheng Deng ◽

Raghavan Chinndurai ◽

Shala Yuan ◽

Andrea Pennati ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Fusion Protein ◽

Nk Cells ◽

Cd8 T Cells ◽

Hematological Malignancies ◽

Granzyme B ◽

Flow Cytometric Analysis ◽

Cd8 T Cell ◽

P Value

Abstract The use of cytokines as agents to augment immune responses against malignancies have been dealt setbacks due to immune selection of tumors, resulting in subpopulations that elaborate tumor-derived soluble factors, such as transforming growth factor-beta (TGF-β), which suppress immune effector functions. TGF-β is overexpressed by many solid and hematological malignancies and is well known to inhibit the proliferation and anti-tumor functions of lymphomyeloid cells. In order to maximize cytokine-based immunotherapy against tumors, we have designed a novel fusion protein consisting of proinflammatory murine interleukin-15 (IL-15) linked to the sushi domain of the IL-15Rα chain (IL15Rαsushi +IL15) fused in frame to the C'-terminus of a dimeric murine TGF-β-receptor (type II, TβRII) ectodomain-based ligand trap, termed FIST-15 (Fusion of Interleukin 15 with Sushi to TGF-β receptor). The rationale for the design of this protein is to prevent tumor-derived TGF-β from suppressing the immune response via the TGF-β ligand trap moiety, while simultaneously providing a potent stimulus for the activation of anti-tumor responses by an IL-15R agonist (IL-15Rαsushi +IL15). FIST-15 can neutralize TGF-β induced Smad signaling, and induce STAT3 and STAT5 phosphorylation by immunoblot and intracellular flow cytometric analysis of lymphocytes, suggesting that both protein domains are biochemically active. Functionally, FIST-15 is able to induce CD8+ T-cell proliferation at rates greater than IL-15 alone (CD8+ T-cell replicative index or fold-expansion of responding cells: 40, FIST-15, vs. 10, IL-15; p-value of unpaired T-test <0.05). The mitogenic effects of IL-15 are abrogated in CD8+ T-cells and NK cells in the presence of TGF-β. However, FIST-15 can overcome TGF-β mediated inhibition in both these cellular subsets (CD8+ T-cell replicative index: 20, FIST-15, vs. 5, IL-15, and NK cell replicative index: 40, FIST-15 vs. 5, IL-15; p-value <0.05). Rapid proliferation of the CD8+ central memory phenotype (CD62L+, CD44+) T-cells are seen with FIST-15 treatment. Compared to IL-15 expanded CD8+ T-cells, FIST-15 treatment also produced more IFN-γ, TNF-α, and IL-2 secreting CD8+ T-cells upon PMA/ionomycin stimulation. In addition to cytokines, production of anti-tumor effector molecules such as granzyme B is known to be inhibited by TGF-β. FIST-15 treated NK cells were superior to IL-15 treated NK cells in granzyme B production, even in the presence of TGF-β, as assayed by flow cytometric analysis (86.8% vs. 30.7% granzyme B expressing cells). Functionally, FIST-15 treated NK cells were also significantly more cytolytic against TGF-β secreting B16 murine melanoma cells in vitro compared to IL-15 treated NK cells (83.5% killing, FIST-15, vs. 24.4% killing, IL-15). C57Bl/6 mice with pre-established, syngeneic B16 melanoma tumors were treated with FIST-15 to assay the anti-tumor effects of the fusion protein in vivo. Mice receiving FIST-15 showed a significant delay in tumor growth (mean tumor volume: 345mm3) compared to control mice receiving conditioned media (mean tumor volume: 814.12mm3; p-value of paired T-test = 0.02) by day 21 post-tumor implantation. Furthermore, FIST-15 treated mice showed a significant survival advantage compared to control treated mice (80% vs 0%; p-value of log rank test = 0.0019) by day 27 post-tumor implantation. Mice immunized with B16 tumors transduced to express FIST-15 were also protected against subsequent wildtype B16 tumor challenge, suggesting that FIST-15 can trigger an adaptive immune response against tumor. Ongoing work utilizing FIST-15 in murine models of hematological malignancies, such as EL-4 lymphoma and C1498 AML, is currently underway. These models were selected due to their known overexpression of TGF-β isoforms that systemically inhibit endogenous anti-tumor responses, as well as the efficacy of immunotherapeutic agents. Indeed, many hematological malignancies acquire mutations that render them insensitive to the growth-inhibitory effects of TGF-β, where it may then be overexpressed as an oncogene to promote further tumor growth by inhibiting the immune system's anti-tumor capabilities (Dong et al Blood 2006). FIST-15 may present a viable immunotherapeutic strategy for hematological malignancies by combining the immune activating effects of IL-15 with the neutralization of immunosuppressive TGF-β. Disclosures Ng: Emory University: Patents & Royalties. Galipeau:Emory University: Patents & Royalties.

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Lactobacillus plantarum surface-displayed ASFV (p54) with porcine IL-21 generally stimulates protective immune responses in mice

AMB Express ◽

10.1186/s13568-021-01275-9 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Xiao-Lei Chen ◽

Jun-Hong Wang ◽

Wei Zhao ◽

Chun-Wei Shi ◽

Kai-Dian Yang ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

Immune Responses ◽

Fusion Protein ◽

Lactobacillus Plantarum ◽

Cd8 T Cells ◽

Classical Swine Fever ◽

Oral Vaccines ◽

Ifn Γ ◽

Mtt Assays

AbstractAfrican classical swine fever virus (ASFV) has spread seriously around the world and has dealt with a heavy blow to the pig breeding industry due to the lack of vaccines. In this study, we produced recombinant Lactobacillus plantarum (L. plantarum) expressing an ASFV p54 and porcine IL-21 (pIL-21) fusion protein and evaluated the immune effect of NC8-pSIP409-pgsA'-p54-pIL-21 in a mouse model. First, we verified that the ASFV p54 protein and p54-pIL-21 fusion protein were anchored on the surface of L. plantarum NC8 by flow cytometry, immunofluorescence and Western blotting. Then, the results were verified by flow cytometry, ELISA and MTT assays. Mouse-specific humoral immunity and mucosal and T cell-mediated immune responses were induced by recombinant L. plantarum. The results of feeding mice recombinant L. plantarum showed that the levels of serum IgG and mucosal secreted IgA (SIgA), the number of CD4 and CD8 T cells, and the expression of IFN-γ in CD4 and CD8 T cells increased significantly, and lymphocyte proliferation occurred under stimulation with the ASFV p54 protein. Our data lay a foundation for the development of oral vaccines against ASFV in the future.

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A soluble divalent class I MHC/IgG1 fusion protein activates CD8+ T cells in vivo

Clinical Immunology ◽

10.1016/j.clim.2005.02.013 ◽

2005 ◽

Vol 116 (1) ◽

pp. 65-76 ◽

Cited By ~ 4

Author(s):

Brenna Carey ◽

Monica DeLay ◽

Jane E. Strasser ◽

Claudia Chalk ◽

Kristen Dudley-McClain ◽

...

Keyword(s):

T Cells ◽

Fusion Protein ◽

Cd8 T Cells ◽

Class I ◽

Class I Mhc

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Natural Killer Cell–Mediated Eradication of Neuroblastoma Metastases to Bone Marrow by Targeted Interleukin-2 Therapy

Blood ◽

10.1182/blood.v91.5.1706.1706_1706_1715 ◽

1998 ◽

Vol 91 (5) ◽

pp. 1706-1715 ◽

Cited By ~ 1

Author(s):

Holger N. Lode ◽

Rong Xiang ◽

Torsten Dreier ◽

Nissi M. Varki ◽

Stephen D. Gillies ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

Fusion Protein ◽

Nk Cells ◽

Natural Killer ◽

Therapeutic Effect ◽

Cd8 T Cells ◽

Nk Cell ◽

Interleukin 2

Targeted interleukin-2 (IL-2) therapy with a genetically engineered antidisialoganglioside GD2 antibody–IL-2 fusion protein induced a cell-mediated antitumor response that effectively eradicated established bone marrow and liver metastases in a syngeneic model of neuroblastoma. The mechanism involved is exclusively natural killer (NK) cell–dependent, because NK-cell deficiency abrogated the antitumor effect. In contrast, the fusion protein remained completely effective in the T-cell–deficient mice or immunocompetent mice depleted of CD8+ T cells in vivo. A strong stimulation of NK-cell activity was also shown in vitro. Immunohistology of the leukocytic infiltrate of livers from treated mice revealed a strong staining for NK cells but not for CD8+ T cells. The therapeutic effect of the fusion protein was increased when combined with NK-cell–stimulating agents, such as poly I:C or recombinant mouse interferon-γ. In conclusion, these data show that targeted delivery of cytokines to the tumor microenvironment offers a new strategy to elicit an effective cellular immune response mediated by NK cells against metastatic neuroblastoma. This therapeutic effect may have general clinical implications for the treatment of patients with minimal residual disease who suffer from T-cell suppression after high-dose chemotherapy but are not deficient in NK cells.

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