IM-6 HVJ-E containing PD-L1 siRNA inhibits immunosuppressive activities and elicits antitumor immune responses in glioma

Abstract Inactivated Sendai virus particle, hemagglutinating virus of Japan-envelope (HVJ-E), is a non-replicating virus-derived vector, in which the genomic RNA of Sendai virus (HVJ) has been destroyed. HVJ-E is a promising vector that enables the highly efficient and safe introduction of enclosed molecules such as RNA into target cells. Moreover, HVJ-E provokes robust antitumoral immunity by activating natural killer (NK) cells and CD8+ T lymphocytes and their induction into the tumor periphery, and by suppressing regulatory T lymphocytes (Treg) locally in the tumor. In the present study, we investigated a novel combination of antitumor immunotherapy by the antitumor immune-activating effect of HVJ-E itself with the inhibition of tumor PD-L1 molecule expression. We confirmed that intratumoral injection of HVJ-E containing siRNA targeting PD-L1 (siPDL1/HVJ-E) inhibited tumor PD-L1 protein expression in a mouse subcutaneous tumor model using TS, a mouse glioma stem-like cell. We conducted treatment experiments in the mouse brain tumor model in three groups: control group (PBS), siNC/HVJ-E group (negative control siRNA + HVJ-E), and siPDL1/HVJ-E group. We obtained a significant prolongation of overall survival in the siPDL1/HVJ-E group. Flow cytometric analyses of brain tumor models showed that the proportions of brain-infiltrating CD8+ T lymphocytes and NK cells were significantly increased after giving siPDL1/HVJ-E; in contrast, the rate of Treg/CD4+ lymphocytes was significantly decreased in HVJ-E-treated tumors (siNC/HVJ-E and siPDL1/HVJ-E). No difference was observed in the proportions of macrophages or M2 macrophages. CD8 depletion abrogated the therapeutic effect of siPDL1/HVJ-E, indicating that CD8+ T lymphocytes mainly mediated this therapeutic effect. We believe that this non-replicating immunovirotherapy may be a novel therapeutic alternative to treat patients with glioblastoma. The full article has been published (Cancer Science. 2021 Jan;112(1):81–90).

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Differential expression of granzymes A and B in human cytotoxic lymphocyte subsets and T regulatory cells

Blood ◽

10.1182/blood-2004-03-0859 ◽

2004 ◽

Vol 104 (9) ◽

pp. 2840-2848 ◽

Cited By ~ 304

Author(s):

William J. Grossman ◽

James W. Verbsky ◽

Benjamin L. Tollefsen ◽

Claudia Kemper ◽

John P. Atkinson ◽

...

Keyword(s):

T Lymphocytes ◽

Nk Cells ◽

T Regulatory Cells ◽

Lymphocyte Subsets ◽

Granzyme B ◽

Human Lymphocytes ◽

Target Cells ◽

Regulatory Cells ◽

Cd8 T Lymphocytes ◽

Tr1 Cells

Abstract Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells use the perforin/granzyme pathway as a major mechanism to kill pathogen-containing cells and tumor cells.1,2 Dysregulation of this pathway results in several human diseases, such as hemophagocytic lymphohistiocytosis. Here we characterize the single-cell expression pattern of granzymes A and B in human lymphocytes using a flow cytometry-based assay. We demonstrate that most circulating CD56+8- NK cells, and approximately half of circulating CD8+ T lymphocytes, coexpressed both granzymes A and B. In contrast, few circulating CD4+ T lymphocytes expressed granzymes A or B. Activation of CD8+ T lymphocytes with concanavalin A (ConA)/interleukin-2 (IL-2), and activation of CD4+ T lymphocytes with antibodies to CD3/CD28 or CD3/CD46 (to generate T regulatory [Tr1] cells), induced substantial expression of granzyme B, but not granzyme A. Naive CD4+CD45RA+ cells stimulated with antibodies to CD3/CD46 strongly expressed granzyme B, while CD3/CD28 stimulation was ineffective. Finally, we show that granzyme B-expressing CD4+ Tr1 cells are capable of killing target cells in a perforin-dependent, but major histocompatibility complex (MHC)/T-cell receptor (TCR)-independent, manner. Our results demonstrate discordant expression of granzymes A and B in human lymphocyte subsets and T regulatory cells, which suggests that different granzymes may play unique roles in immune system responses and regulation.

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N-linked oligosaccharides can protect target cells from the lysis mediated by NK cells but not by cytotoxic T lymphocytes: role of NKG2-A

Tissue Antigens ◽

10.1034/j.1399-0039.1999.540201.x ◽

1999 ◽

Vol 54 (2) ◽

pp. 113-121 ◽

Cited By ~ 4

Author(s):

M.-A. Sol ◽

N. Vacaresse ◽

J. Lule ◽

C. Davrinche ◽

B. Gabriel ◽

...

Keyword(s):

T Lymphocytes ◽

Nk Cells ◽

Cytotoxic T Lymphocytes ◽

Target Cells

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Cord blood T lymphocytes lack constitutive perforin expression in contrast to adult peripheral blood T lymphocytes

Blood ◽

10.1182/blood.v85.6.1540.bloodjournal8561540 ◽

1995 ◽

Vol 85 (6) ◽

pp. 1540-1546 ◽

Cited By ~ 64

Author(s):

C Berthou ◽

S Legros-Maida ◽

A Soulie ◽

A Wargnier ◽

J Guillet ◽

...

Keyword(s):

T Lymphocytes ◽

Cord Blood ◽

Nk Cells ◽

Peripheral Blood ◽

Cell Subset ◽

Cytolytic Activity ◽

Lower Percentage ◽

Target Cells ◽

Perforin Expression ◽

Adult Peripheral Blood

Perforin is the cytolytic pore-forming protein, which alone can be responsible for the lethal hit in one of the killing mechanisms used by natural killer (NK) cells or cytotoxic T lymphocytes. In this study, perforin expression was investigated in cord blood (CB) lymphocytes to determine their killing potential in vivo. The majority of CB CD3- NK cells had the protein. Compared with adult perforin-positive NK cells, a significantly lower percentage of cells expressing CD56 and CD57, the related neural cell adhesion molecules, was found (P = .0001). Perforin was also present in a unique immature CB NK-cell subset, characterized by cytoplasmic CD3 antigen (Ag) expression. In CB, very few CD8 perforin-positive T lymphocytes could be detected, but they were in significant numbers in adult peripheral blood (P = .02). A substantial proportion of these cells (70% +/- 23%) lacked the CD28 T-cell coactivation Ag, and they were able to exert NK-like, major histocompatibility complex nonrestricted cytolytic activity. CD4+ and gamma delta-T cells expressing perforin were absent from CB, but low numbers of such cells were detected in adult peripheral blood (P = .0001). Therefore, the spontaneous cytolytic activity of CB lymphocytes appeared to be dependent on well-represented perforin-positive NK cells, which were shown to efficiently lyse NK-sensitive target cells.

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Improved Immune Recovery after Transplantation of TCRαβ/CD19 Depleted Allografts from Haploidentical Donors in Pediatric Patients

Blood ◽

10.1182/blood.v124.21.852.852 ◽

2014 ◽

Vol 124 (21) ◽

pp. 852-852

Author(s):

Peter Lang ◽

Tobias Feuchtinger ◽

Heiko-Manuel Teltschik ◽

Wolfgang Schwinger ◽

Patrick Schlegel ◽

...

Keyword(s):

Stem Cells ◽

T Cells ◽

T Lymphocytes ◽

Nk Cells ◽

Pediatric Patients ◽

Conflicts Of Interest ◽

Γδ T Cells ◽

Control Group ◽

Unrelated Donor ◽

Immune Recovery

Abstract Transplantation of haploidentical stem cells has become an accepted option for pediatric patients and adults with high risk malignancies who lack a matched related or unrelated donor. In recent years, the majority of pediatric transplant centers chose the CD34 positive selection of peripheral stem cells, which allowed minimizing GvHD by effective reduction of T cells in the graft. However, infectious complications caused by delayed immune recovery were a major reason for transplant related mortality (TRM). In order to improve the immune recovery, we have established a new T-cell depletion method which removes αβ+ T-lymphocytes via a biotinylated anti-TcRαβ antibody followed by an anti-biotin antibody conjugated to magnetic microbeads while retaining γδ+ T-lymphocytes, natural killer (NK) cells and other cells in the graft. In addition, CD19+ B-lymphocytes were concomitantly depleted for the prevention of post-transplant EBV-associated lymphoproliferative disease. Immune recovery was retrospectively analyzed in a cohort of 41 patients with acute leukemia, MDS and non-malignant diseases, who received αβ T and B cell depleted allografts from haploidentical family donors. Conditioning regimens consisted of fludarabine or clofarabine, thiotepa, melphalan and serotherapy with OKT3 or ATG-Fresenius®. Graft manipulation was carried out with anti TCRαβ and anti CD19 antibodies and immunomagnetic microbeads. γδ T cells and NK cells remained in the grafts. Primary engraftment occurred in 88%, acute graft versus host disease (aGvHD) grade II and III-IV occurred in 10% and 15%. Immune recovery data were available in 26 patients and comparable after OKT3 (n=7) or ATG-F® (n=19). Median time to reach > 100 CD3+/µl, > 200 CD19+ cells/µl and > 200 CD56+ cells/µl for the whole group was 13, 127 and 12.5 days. Compared to a historical control group of patients with CD34 positive selected grafts, significantly higher cell numbers were found for CD3+ at days +30 and +90 (267 vs. 27 and 397 vs. 163 cells/µl), for CD3+4+ at day +30 (58 vs. 11 cells/µl) and for CD56+ at day +14 (622 vs. 27 cells/µl). The clinical impact of this accelerated immune recovery will be evaluated in an ongoing prospective multi-center trial. Disclosures No relevant conflicts of interest to declare.

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Interleukin-2 enhances the response of natural killer cells to interleukin-12 through up-regulation of the interleukin-12 receptor and STAT4

Blood ◽

10.1182/blood.v95.10.3183.010k36_3183_3190 ◽

2000 ◽

Vol 95 (10) ◽

pp. 3183-3190 ◽

Cited By ~ 4

Author(s):

Kathy S. Wang ◽

David A. Frank ◽

Jerome Ritz

Keyword(s):

T Lymphocytes ◽

Nk Cells ◽

Natural Killer ◽

Critical Role ◽

Interleukin 2 ◽

Interleukin 12 ◽

Target Cells ◽

Functional Responses ◽

Antitumor Effects ◽

Ifn Γ

Interleukin (IL)-12 plays a critical role in modulating the activities of natural killer (NK) cells and T lymphocytes. In animal models, IL-12 has potent antitumor effects that are likely mediated by its ability to enhance the cytotoxic activity of NK cells and cytotoxic T lymphocytes, and to induce the production of interferon (IFN)-γ by NK and T cells. In addition to IL-12, NK cells are responsive to IL-2, and may mediate some of the antitumor effects of IL-2. In this study, we examine the interaction between IL-2 and the signaling events induced by IL-12 in NK cells. We find that IL-2 not only up-regulates the expression of IL-12Rβ1 and IL-12Rβ2, it also plays an important role in up-regulating and maintaining the expression of STAT4, a critical STAT protein involved in IL-12 signaling in NK cells. In contrast to the effects of IL-2 alone, expression of IL-12 receptors and STAT4 are unaffected or decreased by IL-12 or the combination of IL-2 and IL-12. Through expression of high levels of IL-12 receptors and STAT4, IL-2–primed NK cells show enhanced functional responses to IL-12 as measured by IFN-γ production and the killing of target cells. NK cells from cancer patients who received low-dose IL-2 treatment also exhibited increased expression of IL-12 receptor chains, suggesting that IL-2 may enhance the response to IL-12 in vivo. These findings provide a molecular framework to understand the interaction between IL-2 and IL-12 in NK cells, and suggest strategies for improving the effectiveness of these cytokines in the immunotherapy of cancer.

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Antigen recognition by H-2-restricted cytolytic T lymphocytes is not mediated by two independent receptors.

Journal of Experimental Medicine ◽

10.1084/jem.159.1.330 ◽

1984 ◽

Vol 159 (1) ◽

pp. 330-335 ◽

Cited By ~ 6

Author(s):

D T Harris ◽

H R MacDonald ◽

J C Cerottini

Keyword(s):

T Lymphocytes ◽

Sendai Virus ◽

Leukemia Virus ◽

Antigen Recognition ◽

Target Cells ◽

Cytolytic T Lymphocytes ◽

Antigen Receptors ◽

Nominal Antigen ◽

Moloney Leukemia Virus ◽

Fusion Products

Detergent-solubilized murine cytolytic T lymphocytes (CTL) clones were incorporated into Sendai virus-containing synthetic liposomes. When these liposomes were then fused with other CTL clones possessing a different non-cross-reacting specificity, the fusion products were observed to lyse target cells recognized by both parental CTL clones. This method was then used with two H-2-restricted CTL clones of different, non-cross-reacting specificities (anti-H-2b-H-Y or anti-H-2b Moloney leukemia virus). Once again, the fusion products were found to be lytic against both target cells recognized by the parental clones, but in no instance was there any observable lysis of target cells bearing the same nominal antigen in the context of different H-2 molecules. These results provide strong evidence that antigen recognition by H-2-restricted CTL is not mediated by two independent antigen receptors.

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Polymorphisms in the IFNγ, IL-10, and TGFβGenes May Be Associated with HIV-1 Infection

Disease Markers ◽

10.1155/2015/248571 ◽

2015 ◽

Vol 2015 ◽

pp. 1-9 ◽

Cited By ~ 5

Author(s):

Felipe Bonfim Freitas ◽

Sandra Souza Lima ◽

Rosimar Neris Martins Feitosa ◽

Vânia Nakauth Azevedo ◽

Marluísa de O. Guimarães Ishak ◽

...

Keyword(s):

Viral Load ◽

T Lymphocytes ◽

Plasma Viral Load ◽

Serum Levels ◽

High Serum ◽

Control Group ◽

Clinical Markers ◽

Cd8 T Lymphocytes ◽

Aids Progression ◽

Hiv 1

Objective.This study investigated possible associations between the TNFα-308G/A, IFN+874A/T, IL-6-174C/G, IL-10-1082A/G, and TGFβ-509C/T polymorphisms with HIV-1 infection, in addition to correlation of the polymorphisms with clinical markers of AIDS progression, such as levels of CD4+/CD8+ T lymphocytes and plasma viral load.Methods.A total of 216 individuals who were infected with HIV-1 and on antiretroviral therapy (ART) and 294 individuals from the uninfected control group were analyzed.Results.All individuals evaluated were negative for total anti-HBc, anti-HCV, anti-T. pallidum, and anti-HTLV-1/2. The polymorphisms were identified by PCR-RFLP. Individuals presenting the IFN+874A allele as well as the AA genotype were more frequent in the HIV-1 infected group compared to the control group (P<0.05), in addition to having lower levels of CD4+ T lymphocytes. The CD8+ T lymphocytes count was significantly lower in individuals with the IL-10-1082 GG genotype. The TGFβ-509TT genotype was associated with higher plasma viral load.Conclusions.The results suggest that the presence of the IFN+874A allele confers susceptibility to HIV-1 infection and a decrease in the number of CD4+ T lymphocytes. In addition, the genotype associated with high serum levels of TGFβmay be associated with an increase in plasma viral load.

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Immune Profile of the Normal Maternal-Fetal Interface in Rhesus Macaques and Its Alteration Following Zika Virus Infection

Frontiers in Immunology ◽

10.3389/fimmu.2021.719810 ◽

2021 ◽

Vol 12 ◽

Author(s):

Matilda J. Moström ◽

Elizabeth A. Scheef ◽

Lesli M. Sprehe ◽

Dawn Szeltner ◽

Dollnovan Tran ◽

...

Keyword(s):

T Lymphocytes ◽

Nk Cells ◽

B Lymphocytes ◽

Vertical Transmission ◽

Zika Virus ◽

Peripheral Blood ◽

Congenital Infection ◽

Effector Memory ◽

Cd8 T Lymphocytes ◽

Maternal Fetal Interface

The maternal decidua is an immunologically complex environment that balances maintenance of immune tolerance to fetal paternal antigens with protection of the fetus against vertical transmission of maternal pathogens. To better understand host immune determinants of congenital infection at the maternal-fetal tissue interface, we performed a comparative analysis of innate and adaptive immune cell subsets in the peripheral blood and decidua of healthy rhesus macaque pregnancies across all trimesters of gestation and determined changes after Zika virus (ZIKV) infection. Using one 28-color and one 18-color polychromatic flow cytometry panel we simultaneously analyzed the frequency, phenotype, activation status and trafficking properties of αβ T, γδ T, iNKT, regulatory T (Treg), NK cells, B lymphocytes, monocytes, macrophages, and dendritic cells (DC). Decidual leukocytes showed a striking enrichment of activated effector memory and tissue-resident memory CD4+ and CD8+ T lymphocytes, CD4+ Tregs, CD56+ NK cells, CD14+CD16+ monocytes, CD206+ tissue-resident macrophages, and a paucity of B lymphocytes when compared to peripheral blood. t-distributed stochastic neighbor embedding (tSNE) revealed unique populations of decidual NK, T, DC and monocyte/macrophage subsets. Principal component analysis showed distinct spatial localization of decidual and circulating leukocytes contributed by NK and CD8+ T lymphocytes, and separation of decidua based on gestational age contributed by memory CD4+ and CD8+ T lymphocytes. Decidua from 10 ZIKV-infected dams obtained 16-56 days post infection at third (n=9) or second (n=1) trimester showed a significant reduction in frequency of activated, CXCR3+, and/or Granzyme B+ memory CD4+ and CD8+ T lymphocytes and γδ T compared to normal decidua. These data suggest that ZIKV induces local immunosuppression with reduced immune recruitment and impaired cytotoxicity. Our study adds to the immune characterization of the maternal-fetal interface in a translational nonhuman primate model of congenital infection and provides novel insight in to putative mechanisms of vertical transmission.

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Features of laboratory markers in pregnant women with antiphospholipid syndrome and retrochorial hematoma

EUREKA Health Sciences ◽

10.21303/2504-5679.2021.001973 ◽

2021 ◽

pp. 12-19

Author(s):

Oleh Tomniuk

Keyword(s):

Platelet Aggregation ◽

T Lymphocytes ◽

Pregnant Women ◽

Nk Cells ◽

Antiphospholipid Syndrome ◽

Annexin V ◽

Main Group ◽

Control Group ◽

Antiphospholipid Antibody ◽

Willebrand Factor

The aim. Study of hemostasis, antiphospholipid antibody levels and immunological parameters in pregnant women with antiphospholipid syndrome (APS), in particular with retrochorial hematoma (RCH). Materials and methods. 90 women were selected and divided into two groups: the control group – 30 pregnant women with a normal pregnancy (without APS) and the main group – 60 pregnant women with APS. Women in the main group were diagnosed with APS before pregnancy. In turn, the main group was divided into two subgroups: 1 subgroup – 41 pregnant women without RCH and 2 subgroup – 19 pregnant women with RCH. The main indicators of hemostasis were determined in all pregnant women, namely: the degree and rate of platelet aggregation, Willebrand factor, D-dimers. In addition, the level of antiphospholipid antibodies (APLA), antibodies to β2-glycoprotein, to annexin V, to prothrombin was examined, and the level of annexin V was also determined. The absolute and relative content of Treg, CD3+, T-lymphocytes, CD4+ (T-helpers), CD8+ (cytotoxic T-lymphocytes), CD19+ (B-lymphocytes), CD16+CD56+ (NK cells), CD16+CD56+CD107a+ (activated NK cells). Results. The obtained results showed that in pregnant women with APS compared to pregnant women without APS there are statistically significantly higher values of the degree and rate of platelet aggregation (90.6±6.3% and 106.3±6.7% vs. 65.3±5.3 % and 73.4±5.6%, respectively). There were also higher values of Willebrand factor and D-dimers (2.5±0.3 IU/ml and 378.1±34.3 ng/ml against 1.7±0.2 IU/ml and 268.1±27, 3 ng/ml, respectively). APLAs were significantly higher in pregnant women with APS compared with pregnant women in the control group, namely: 16.1±1.5 vs. 3.8±0.4 U/ml. With regard to antibodies to β2-glycoprotein, to annexin V, to prothrombin and to the level of annexin V, their values were also statistically significantly higher in the group of pregnant women with APS. In addition, the results of the study showed that pregnant women with APS showed changes in subpopulations of immunocompetent cells. However, examining the difference in hemostasis, antibody content, and level of lymphocyte subpopulations between pregnant women with APS with and without RCH, it was found that their shifts in pregnant women with RCH were more pronounced than in women without RCH. Conclusions. Pregnant women with APS are characterized by significantly more significant changes in hemostasis, manifested by activation of intravascular thrombosis. In addition, such pregnant women had a significantly higher concentration of autoantibodies. There are also changes in the immune system, in particular, a decrease in Treg-cells, which have the ability to reduce the specific proliferation and effector functions of lymphocytes, thereby participating in the pathogenesis of APS.

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