Search

Introduction: T-lymphopenia (TLP) is a frequently observed condition in cancer patients, often exacerbated by conventional chemo/radiotherapy, which impairs the efficacy of subsequent immune checkpoint blockade (ICB) therapy. This study aimed to understand the impact of TLP on ICB responsiveness and explore potential therapeutic strategies to enhance antitumor immunity. Methods: To investigate ICB responsiveness depending on the severity of TLP, first, we established TLP mouse models that mimic clinically observed mild and severe TLP through thymectomy and anti-Thy1-induced peripheral T cell depletion. T cell-replete mice and T-lymphopenic mice were inoculated with palpable or advanced tumors to evaluate the antitumor efficacy of anti-PD-1 therapy according to the severity of TLP. Additionally, by utilizing established murine models, we analyzed matched blood, tumor-draining lymph nodes (TDLNs), and tumor samples by flow cytometry to investigate the mechanisms by which ICB responsiveness is impaired under T-lymphopenic conditions. Finally, to evaluate the combination effect of anti-PD-1 and recombinant IL-7 cytokine therapy (rhIL-7-hyFc) in T-lymphopenic conditions, we administered anti-PD-1, rhIL-7-hyFc, or both to advanced tumor-bearing T-lymphopenic mice and subsequently analyzed tumor growth and survival rates. Results: Using mouse models mimicking clinical TLP, we observed that the antitumor efficacy of anti-PD-1 therapy was severely impaired in TLP, depending on the degree of TLP and the immunogenicity of the tumors. TLP mice showed a significant reduction in systemic CD8 T cells but stable intratumoral CD8 T cell numbers, suggesting maintained tumor infiltration despite systemic downregulation. Crucially, TLP led to a shift in the composition of tumor-infiltrating lymphocytes, with a decrease in PD-1+ tumor-reactive CD8 T cells and an increase in PD-1− bystander cells. This reduction in PD-1+ cells was linked to impaired clonal expansion in tumor-draining lymph nodes. To counteract these effects, we introduced recombinant IL-7 cytokine therapy (rhIL-7-hyFc), which effectively restored systemic T cell counts, enhanced PD-1+ CD8 T cell proliferation within tumors, and increased the population of stem-like progenitor cells. The combination of rhIL-7-hyFc and anti-PD-1 therapy resulted in significant tumor regression and improved mouse survival. Discussion: Our findings highlight the critical role of IL-7 in reshaping the CD8 T cell landscape to improve ICB efficacy in TLP conditions, proposing a sequential therapeutic approach: conventional therapy to reduce tumor burden and enhance immunogenicity, followed by IL-7 therapy to restore and rejuvenate CD8 T cells, culminating in effective ICB treatment. [ABSTRACT FROM AUTHOR]

A low lymphocyte count puts immune‐compromised patients at risk of mortality. hIL‐7‐hyFc is a homodimeric interleukin‐7 (IL‐7), a potent T‐cell amplifier, fused to the hybridizing IgD/IgG4 immunoglobulin domain. We performed a randomized, double‐blind, placebo‐controlled, dose‐escalation, phase I study to assess the pharmacokinetic, pharmacodynamic, safety, tolerability, and immunogenicity profiles of hIL‐7‐hyFc administered s.c. and i.m. to healthy volunteers. Thirty subjects randomly received hIL‐7‐hyFc or its matching placebo in an 8:2 ratio at 20, 60 μg/kg s.c., or 60 μg/kg i.m. The hIL‐7‐hyFc was slowly absorbed and its terminal half‐life was 63.26 hours after i.m. administration. The hIL‐7‐hyFc increased absolute lymphocyte count, mostly in T‐cells, which peaked 3 weeks after administration and then lasted for several additional weeks. The hIL‐7‐hyFc was well‐tolerated after a single s.c. and i.m. administration. Injection site reaction was the most common treatment‐emergent adverse event, which resolved spontaneously without treatment. The hIL‐7‐hyFc can be developed into a beneficial treatment option for patients with compromised T‐cell immunity. This trial was registered at www.clinicaltrials.gov as #NCT02860715. [ABSTRACT FROM AUTHOR]

Introduction: T-lymphopenia (TLP) is a frequently observed condition in cancer patients, often exacerbated by conventional chemo/radiotherapy, which impairs the efficacy of subsequent immune checkpoint blockade (ICB) therapy. This study aimed to understand the impact of TLP on ICB responsiveness and explore potential therapeutic strategies to enhance antitumor immunity., Methods: To investigate ICB responsiveness depending on the severity of TLP, first, we established TLP mouse models that mimic clinically observed mild and severe TLP through thymectomy and anti-Thy1-induced peripheral T cell depletion. T cell-replete mice and T-lymphopenic mice were inoculated with palpable or advanced tumors to evaluate the antitumor efficacy of anti-PD-1 therapy according to the severity of TLP. Additionally, by utilizing established murine models, we analyzed matched blood, tumor-draining lymph nodes (TDLNs), and tumor samples by flow cytometry to investigate the mechanisms by which ICB responsiveness is impaired under T-lymphopenic conditions. Finally, to evaluate the combination effect of anti-PD-1 and recombinant IL-7 cytokine therapy (rhIL-7-hyFc) in T-lymphopenic conditions, we administered anti-PD-1, rhIL-7-hyFc, or both to advanced tumor-bearing T-lymphopenic mice and subsequently analyzed tumor growth and survival rates., Results: Using mouse models mimicking clinical TLP, we observed that the antitumor efficacy of anti-PD-1 therapy was severely impaired in TLP, depending on the degree of TLP and the immunogenicity of the tumors. TLP mice showed a significant reduction in systemic CD8 T cells but stable intratumoral CD8 T cell numbers, suggesting maintained tumor infiltration despite systemic downregulation. Crucially, TLP led to a shift in the composition of tumor-infiltrating lymphocytes, with a decrease in PD-1 + tumor-reactive CD8 T cells and an increase in PD-1 - bystander cells. This reduction in PD-1 + cells was linked to impaired clonal expansion in tumor-draining lymph nodes. To counteract these effects, we introduced recombinant IL-7 cytokine therapy (rhIL-7-hyFc), which effectively restored systemic T cell counts, enhanced PD-1 + CD8 T cell proliferation within tumors, and increased the population of stem-like progenitor cells. The combination of rhIL-7-hyFc and anti-PD-1 therapy resulted in significant tumor regression and improved mouse survival., Discussion: Our findings highlight the critical role of IL-7 in reshaping the CD8 T cell landscape to improve ICB efficacy in TLP conditions, proposing a sequential therapeutic approach: conventional therapy to reduce tumor burden and enhance immunogenicity, followed by IL-7 therapy to restore and rejuvenate CD8 T cells, culminating in effective ICB treatment., Competing Interests: DC, S-KI and SY are employees of the Research Institute of NeoImmuneTech, Inc., which supports the supply of rhIL-7-hyFc and its formulation buffer. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Kang, Choi, Moon, Lee, Oh, Yang, Jeong, Park, Im, Yang and Lee.)

This paper presents an arterial distension monitoring scheme using a field-programmable gate array (FPGA)-based inference machine in an ultrasound scanner circuit system. An arterial distension monitoring requires a precise positioning of an ultrasound probe on an artery as a prerequisite. The proposed arterial distension monitoring scheme is based on a finite state machine that incorporates sequential support vector machines (SVMs) to assist in both coarse and fine adjustments of probe position. The SVMs sequentially perform recognitions of ultrasonic A-mode echo pattern for a human carotid artery. By employing sequential SVMs in combination with convolution and average pooling, the number of features for the inference machine is significantly reduced, resulting in less utilization of hardware resources in FPGA. The proposed arterial distension monitoring scheme was implemented in an FPGA (Artix7) with a resource utilization percentage less than 9.3%. To demonstrate the proposed scheme, we implemented a customized ultrasound scanner consisting of a single-element transducer, an FPGA, and analog interface circuits with discrete chips. In measurements, we set virtual coordinates on a human neck for 9 human subjects. The achieved accuracy of probe positioning inference is 88%, and the Pearson coefficient (r) of arterial distension estimation is 0.838.

Purpose: Patients with glioblastoma (GBM) are treated with radiotherapy (RT) and temozolomide (TMZ). These treatments may cause prolonged systemic lymphopenia, which itself is associated with poor outcomes. NT-I7 is a long-acting IL7 that expands CD4 and CD8 T-cell numbers in humans and mice. We tested whether NT-I7 prevents systemic lymphopenia and improves survival in mouse models of GBM., Experimental Design: C57BL/6 mice bearing intracranial tumors (GL261 or CT2A) were treated with RT (1.8 Gy/day × 5 days), TMZ (33 mg/kg/day × 5 days), and/or NT-I7 (10 mg/kg on the final day of RT). We followed the mice for survival while serially analyzing levels of circulating T lymphocytes. We assessed regulatory T cells (Treg) and cytotoxic T lymphocytes in the tumor microenvironment, cervical lymph nodes, spleen, and thymus, and hematopoietic stem and progenitor cells in the bone marrow., Results: GBM tumor-bearing mice treated with RT+NT-I7 increased T lymphocytes in the lymph nodes, thymus, and spleen, enhanced IFNγ production, and decreased Tregs in the tumor which was associated with a significant increase in survival. NT-I7 also enhanced central memory and effector memory CD8 T cells in lymphoid organs and tumor. Depleting CD8 T cells abrogated the effects of NT-I7. Furthermore, NT-I7 treatment decreased progenitor cells in the bone marrow., Conclusions: In orthotopic glioma-bearing mice, NT-I7 mitigates RT-related lymphopenia, increases cytotoxic CD8 T lymphocytes systemically and in the tumor, and improves survival. A phase I/II trial to evaluate NT-I7 in patients with high-grade gliomas is ongoing (NCT03687957)., (©2022 American Association for Cancer Research.)

IL-23 is the key cytokine that induces the expansion of Th17 cells. It is composed of p19 and p40 subunits of IL-12. The p40 subunit binds competitively to the receptor of IL-23 and blocks its activity. Our aim was to assess the preventive and therapeutic effect of the IL-12p40 homodimer (p40)2 subunit in autoimmune arthritis animal models. In the current study, using IL-1R antagonist-knockout mice and a collagen-induced arthritis model, we investigated the suppressive effect of (p40)2 on inflammatory arthritis. We demonstrated that the recombinant adenovirus-expressing mouse (p40)2 model prevented the development of arthritis when given before the onset of arthritis. It also decreased the arthritis index and joint erosions in the mouse model if transferred after arthritis was established. (p40)2 inhibited the production of inflammatory cytokines and Ag-specific T cell proliferation. It also induced CD4(+)CD25(+)Foxp3 regulatory T (Treg) cells in vitro and in vivo, whereas the generation of retinoic acid receptor-related organ receptor γt and Th17 cells was suppressed. The induction of Treg cells and the suppression of Th17 cells were mediated via activated STAT5 and suppressed STAT3. Our data suggest that (p40)2 suppressed inflammatory arthritis successfully. This could be a useful therapeutic approach in autoimmune arthritis to regulate the Th17/Treg balance and IL-23 signaling., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Unlabelled: T follicular helper (Tfh) cells are specialized providers of cognate B cell help, which is important in promoting the induction of high-affinity antibody production in germinal centers (GCs). Interleukin-6 (IL-6) and IL-21 have been known to play important roles in Tfh cell differentiation. Here, we demonstrate that IL-7 plays a pivotal role in Tfh generation and GC formation in vivo, as treatment with anti-IL-7 neutralizing antibody markedly impaired the development of Tfh cells and IgG responses. Moreover, codelivery of mouse Fc-fused IL-7 (IL-7-mFc) with a vaccine enhanced the generation of GC B cells as well as Tfh cells but not other lineages of T helper cells, including Th1, Th2, and Th17 cells. Interestingly, a 6-fold-lower dose of an influenza virus vaccine codelivered with Fc-fused IL-7 induced higher antigen-specific and cross-reactive IgG titers than the vaccine alone in both mice and monkeys and led to markedly enhanced protection against heterologous influenza virus challenge in mice. Enhanced generation of Tfh cells by IL-7-mFc treatment was not significantly affected by the neutralization of IL-6 and IL-21, indicating an independent role of IL-7 on Tfh differentiation. Thus, IL-7 holds promise as a critical cytokine for selectively inducing Tfh cell generation and enhancing protective IgG responses., Importance: Here, we demonstrate for the first time that codelivery of Fc-fused IL-7 significantly increased influenza virus vaccine-induced antibody responses, accompanied by robust expansion of Tfh cells and GC B cells as well as enhanced GC formation. Furthermore, IL-7-mFc induced earlier and cross-reactive IgG responses, leading to striking protection against heterologous influenza virus challenge. These results suggest that Fc-fused IL-7 could be used for inducing strong and cross-protective humoral immunity against highly mutable viruses, such as HIV and hepatitis C virus, as well as influenza viruses., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Dendritic cells (DC) present α-galactosylceramide (αGalCer) to invariant T-cell receptor-expressing natural killer T cells (iNKT) activating these cells to secrete a variety of cytokines, which in turn results in DC maturation and activation of other cell types, including NK cells, B cells, and conventional T cells. In this study, we showed that αGalCer-pulsing of antigen-activated CD8 T cells before adoptive transfer to tumor-bearing mice caused a marked increase in donor T-cell proliferation, precursor frequency, and cytotoxic lymphocyte activity. This effect was interleukin (IL)-2 dependent and involved both natural killer T cells (NKT) and DCs, as mice lacking IL-2, NKTs, and DCs lacked any enhanced response to adoptively transferred αGalCer-loaded CD8 T cells. iNKT activation was mediated by transfer of αGalCer from the cell membrane of the donor CD8 T cells onto the αGalCer receptor CD1d which is present on host DCs. αGalCer transfer was increased by prior activation of the donor CD8 T cells and required AP-2-mediated endocytosis by host DCs. In addition, host iNKT cell activation led to strong IL-2 synthesis, thereby increasing expansion and differentiation of donor CD8 T cells. Transfer of these cells led to improved therapeutic efficacy against established solid tumors in mice. Thus, our findings illustrate how αGalCer loading of CD8 T cells after antigen activation in vitro may leverage the therapeutic potential of adoptive T-cell therapies.

Human IgG1 Fc has been widely used as a bioconjugate, but exhibits shortcomings, such as antibody- and complement-mediated cytotoxicity as well as decreased bioactivity, when applied to agonistic proteins. Here, we constructed a nonimmunogenic, noncytolytic and flexible hybrid Fc (hyFc) consisting of IgD and IgG4, and tested its function using erythropoietin (EPO) conjugate, EPO-hyFc. Despite low amino acid homology (20.5%) between IgD Fc and IgG4 Fc, EPO-hyFc retained "Y-shaped" structure and repeated intravenous administrations of EPO-hyFc into monkeys did not generate EPO-hyFc-specific antibody responses. Furthermore, EPO-hyFc could not bind to FcγR I and C1q in contrast to EPO-IgG1 Fc. In addition, EPO-hyFc exhibited better in vitro bioactivity and in vivo bioactivity in rats than EPO-IgG1 Fc, presumably due to the high flexibility of IgD. Moreover, the mean serum half-life of EPO-hyFc(H), a high sialic acid content form of EPO-hyFc, was approximately 2-fold longer than that of the heavily glycosylated EPO, darbepoetin alfa, in rats. More importantly, subcutaneous injection of EPO-hyFc(H) not only induced a significantly greater elevation of serum hemoglobin levels than darbepoetin alfa in both normal rats and cisplatin-induced anemic rats, but also displayed a delayed time to maximal serum level and twice final area-under-the-curve (AUC(last)). Taken together, hyFc might be a more attractive Fc conjugate for agonistic proteins/peptides than IgG1 Fc due to its capability to elongate their half-lives without inducing host effector functions and hindering bioactivity of fused molecules. Additionally, a head-to-head comparison demonstrated that hyFc-fusion strategy more effectively improved the in vivo bioactivity of EPO than the hyperglycosylation approach.

Background: We previously reported that IFN-gamma producing T cell responses induced by the combined therapy of DNA vaccine and lamivudine for one year are important for the induction of sustained virological response (SVR). However, IFN-gamma production is not sufficient to predict sustained viremia control in chronic hepatitis B (CHB) carriers treated., Methods: Twelve CHB carriers were intramuscularly immunized 12 times at a 4-week interval with 8 mg of HBV DNA vaccine during the standard lamivudine treatment (100 mg/daily/1 year). The level of cytokines during and after the combined therapy in plasma of all 12 CHB carriers treated was determined by each ELISA kit. Six out of 12 CHB carriers revisited the clinic, and their HBV DNA levels were examined., Results: The combined therapy increased plasma IL-12 and IL-12/p40 ratio during the treatment (baseline vs. peak level: 41.8+/-8.3 vs. 163.1+/-29.2 pg/ml; p<0.01 and 0.96+/-0.25 vs. 3.58+/-0.86; p<0.01, espectively), and the peak level of plasma IL-12 and IL-12/p40 ratio was evoked at 6 to 10 months during the combined therapy. In particular, CHB carriers with SVR had two and three-fold higher level of the peak plasma IL-12 and plasma IL-12/p40 ratio than non-virological responders (NVRs), respectively (218.0+/-41.4 vs. 108.1+/-28.6 pg/ml; p=0.09 and 5.35+/-1.38 vs. 1.80+/-0.29; p<0.05, respectively), while p40 level was consistent during the combined therapy. In addition, there was no significant temporal correlation between the peak IL-12/p40 ratio and the elevation of serum alanine aminotransferase (ALT) in this study, contrast to IFN-alpha therapy which induced peak IL-12 level following ALT flares., Conclusion: Our results indicate that the combined therapy induces the increase of plasma IL-12 and IL-12/p40 ratio, which are associated with long-term SVR in CHB carriers.

Pulse-induced permeabilization of cellular membranes, generally referred to as electroporation (EP), has been used for years as a tool to increase macromolecule uptake in tissues, including nucleic acids, for gene therapeutic applications, and this technique has been shown to result in improved immunogenicity. In this study, we assessed the utility of EP as a tool to improve the efficacy of HB-110, a novel therapeutic DNA vaccine against chronic hepatitis B, now in phase 1 of clinical study in South Korea. The potency of HB-110 in mice was shown to be improved by EP. The rapid onset of antigen expression and higher magnitude of humoral and cellular responses in electric pulse-treated mice revealed that EP may enable a substantial reduction in the dosage of DNA vaccine required to elicit a response similar in magnitude to that achievable via conventional administration. This study also showed that EP-based vaccination at 4-week-intervals elicited a cellular immune response which was about two-fold higher than the response elicited by conventional vaccination at 2-week intervals. These results may provide a rationale to reduce the clinical dose and increase the interval between the doses in the multidose vaccination schedule. Electric pulsing also elicited a more balanced immune response against four antigens expressed by HB-110: S, preS, Core, and Pol.

Liver cirrhosis is one of the major complications of hepatitis C virus (HCV) infection, but the mechanisms underlying HCV-related fibrogenesis are still not clear. Although the roles of HCV core protein remain poorly understood, it is supposed to play an important role in the regulation of cellular growth and hepatocarcinogenesis. The aim of this study was to examine the role of HCV core protein on the hepatic fibrogenesis. We established an in vitro co-culture system with primary hepatic stellate cell (HSC) isolated from rats, and a stable HepG2-HCV core cell line which had been transfected with HCV core gene. The expressions of fibrosis-related molecules transforming growth factor beta1 (TGF-beta1), transforming growth factor beta receptor II (TGFbetaRII), alpha-smooth muscle actin (alpha-SMA) and connective tissue growth factor (CTGF) were analyzed via histological or molecular methods. In addition, the expression levels of matrix metaloprotinase-2 (MMP-2) and collagen type I (Col I) from the co-cultured media were measured by zymogram and ELISA, respectively. The expressions of alpha-SMA, TGF-beta1, Col I, TGFbetaRII and MMP-2 were significantly increased in the co-culture of stable HepG2-HCV core with HSC. Moreover, the significant increases of CTGF and TGF-beta1 in the HCV core-expressing cells were observed by either Northern or Western blot analysis. These results suggest that HCV core protein may contribute to the hepatic fibrogenesis via up-regulation of CTGF and TGF-beta1.

Background/aims: The study of liver fibrogenesis by hepatitis C virus (HCV) has been limited due to the lack of an efficiency in vitro culture systems. In the present study, we investigated whether or not HCV core protein is directly related to liver fibrogenesis through stimulation of hepatic stellate cells (HSC)., Methods: Human and rat HSC were isolated and we established an in vitro co-culture system of a stable HepG2-HCV core cell line which was transfected with HCV core gene and primary HSC. We performed immunocytochemical staining and Western and Northern blot analysis in the stimulated HSC by HCV core protein to identify the expression of transforming growth factor beta1 (TGF-beta1), transforming growth factor beta receptor II (TGFbeta R II), alpha-smooth muscle actin (alpha-SMA) and connective tissue growth factor (CTGF). The expression of matrix metalloproteinase-2 (MMP-2) and collagen type I (Col I) in the culture media were measured by zymogram and ELISA, respectively., Results: The expression of TGF-beta1 and CTGF was significantly higher in the stable HepG2-HCV core cell line than in HepG2 cells. Furthermore, the makers related to fibrosis such as alpha-SMA, TGF-beta1, Col I, TGFRII and MMP-2 were highly expressed in the co-culture of stable HepG2-HCV core with HSC., Conclusions: HCV core protein may play a direct role in the fibrogenesis of chronic liver disease with HCV infection.