Differential expression of granzymes A and B in human cytotoxic lymphocyte subsets and T regulatory 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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Low Percent of Granzyme B Positive T-Regulatory Cells (CD4+CD25high) As a Predictor of Acute Graft-Versus-Host Disease (GVHD) after Allogeneic Hematopoietic Stem Cell Transplantation (allo-HSCT)

Blood ◽

10.1182/blood.v124.21.5871.5871 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5871-5871

Author(s):

Mikhail Drokov ◽

Elena N. Parovichnikova ◽

Larisa A Kuzmina ◽

Irina Galtseva ◽

Vera Vasilieva ◽

...

Keyword(s):

T Regulatory Cells ◽

Conflicts Of Interest ◽

Granzyme B ◽

Geometric Mean ◽

Target Cells ◽

Unrelated Donor ◽

Regulatory Cells ◽

Cd4 Cells ◽

Becton Dickinson ◽

Hematopoietic Stem

Abstract Introduction. Granzyme B is a serine protease commonly found in the granules of cytotoxic lymphocytes and natural killer cells. It is secreted with the pore forming protein perforin and mediates apoptosis in target cells. Granzyme B – mediated cytolysis is one of the regulatory mechanisms (together with IL-2 receptors (CD25)) by which T-regulatory cells (Tregs) influence on T-effectors cells. It is a well-known fact that Tregs participate in a pathogenesis of aGVHD and their amount after allo-HSCT inversely correlates with the probability of aGVHD incidence. No clinical data exist regarding the Granzyme B positive Tregs in this setting. Patients and methods. Peripheral blood samples were collected in EDTA-tubes at day +30, +60, +90 after allo-HSCT and at day of acute GVHD onset from 27 patients with hematological malignancies (AML=12, ALL n=12, LPD=2, MDS =1; 6 with active disease, 21 - in CR) after allo-HSCT (from matched unrelated donor n=23, from matched related donor n=4; MAC = 10, RIC=17). The anti-CD4-APC-Cy7, anti-CD25-FITC and anti-Granzyme B-PE (Becton Dickinson, USA) antibodies were used to determine T-regulatory cells population as CD4+CD25high. We did not include anti-FoxP3 antibodies as FoxP3 is not so specific in humans and particularly after allo-HSCT due to technical difficulties, several isoforms, FoxP3-positivity on activated nonregulatory CD4+ cells. CD4- lymphocytes (CD8+ and NK-cells certainly containing granzyme B) were used as internal positive control to define the percent of CD4+CD25highGranzymeB+ cells and gMFI (geometric mean fluorescence intensity) among the mononuclear cell fraction. 50000 of CD4+ cells were analyzed on a BD FACSCanto II (Becton Dickinson, USA). Results. 11 patients (42%) have developed aGVHD (II-IV) at a median time of +26 day (8 - 88) after HSCT. The percent of CD4+CD25highGranzymeB+ cells among CD4+cells at day +30 after allo-HSCT in patients who never developed aGVHD was statistically higher (9,49±2,79; p=0.003) than in patients at day of aGVHD onset (3,8±1,78). It’s worth to note that in 4 patients who did not have signs of a GVHD on day +30 but developed it later (on day +40, +45, +74, +88), the percent of CD4+CD25highGranzymeB+ cells at day +30 was as low (3,38±1,47) as at day of aGVHD onset and significantly lower (p=0.003) than in patients who never developed aGVHD, thus predicting the occurrence of aGVHD in the future. Conclusion Despite the fact that the analyzed group is small, we suggest that low relative amount of CD4+CD25highGranzymeB+ cells after allo-HSCT may contribute to the development aGVHD and even predict it onset. This fact, of course, needs further investigation. Disclosures No relevant conflicts of interest to declare.

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

Neuro-Oncology Advances ◽

10.1093/noajnl/vdab159.028 ◽

2021 ◽

Vol 3 (Supplement_6) ◽

pp. vi7-vi8

Author(s):

Narushi Sugii ◽

Masahide Matsuda ◽

Genki Okumura ◽

Akira Shibuya ◽

Eiichi Ishikawa

Keyword(s):

Brain Tumor ◽

T Lymphocytes ◽

Nk Cells ◽

Therapeutic Effect ◽

Sendai Virus ◽

Tumor Model ◽

Control Group ◽

Target Cells ◽

Cd8 T Lymphocytes ◽

Significant Prolongation

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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Imatinib Inhibits Both CD4+ T Regulatory Cells and CD8+ T Lymphocytes Specifically Directed Against the Leukemia-Associated Antigen RHAMM/CD168.

Blood ◽

10.1182/blood.v108.11.2201.2201 ◽

2006 ◽

Vol 108 (11) ◽

pp. 2201-2201

Author(s):

Jinfei Chen ◽

Anita Schmitt ◽

Baoan Chen ◽

Markus Rojewski ◽

Jochen Greiner ◽

...

Keyword(s):

T Cells ◽

T Lymphocytes ◽

T Regulatory Cells ◽

Residual Disease ◽

Cell Epitope ◽

Myelogenous Leukemia ◽

Lymphocyte Function ◽

Regulatory Cells ◽

Color Flow ◽

Cd8 T Lymphocytes

Abstract Imatinib mesylate (IM, STI571, Gleevec®) is highly effective in the treatment of patients with chronic myelogenous leukemia (CML). In patients with residual disease or relapse after allogeneic stem cell transplantation (allo-SCT), the optimal strategy is a matter of debate: donor lymphocyte infusions (DLIs) alone, IM alone or a combination of both have been administered to CML patients in this clinical situation. As an impairment of anti-viral CD8+ T lymphocyte function by IM has been described, we questioned whether IM also affects specific anti-leukemic CD8+ T lymphocytes. Moreover, we asked to which extent CD4+CD25hi T regulatory cells might be affected by IM thus changing the balance between cytotoxic and tolerogenic T cells. After mixed lymphocyte peptide culture (MLPC), we assessed CD8+ T cells from healthy donors and patients with CML after allo-SCT by tetramer staining/multi-color flow cytometry and enzyme linked immunosorbent spot (ELISPOT) assays, as well as CD4+CD25hicells after 3 days of culture with anti-CD3 and anti-CD28. The release of interferon gamma and granzyme B by CD8+ T lymphocytes specific for R3 (ILSLELMKL), a T cell epitope peptide derived from the leukemia-associated antigen designated RHAMM/CD168 (receptor for hyaluronic acid mediated motility) was inhibited by IM in a clearly dose-dependent fashion in a range from 1 to 25 μM. These T cells were further characterized as CD8+ HLA-A2/R3_tetramer+ WT1_tetra-CD45RA+CD27-CD28-CCR7- effector T cells with the ability to lyse R3 peptide labeled T2 cells and CD34+ CML progenitor cells. The inhibition of CD8+ T lymphocytes was a function of the time of exposure to IM and reversible after removal of IM from the MLPC. On the other hand, the proliferation and function of CD4+CD25hiFoxP3+GITR+TGFß1+ CD69+CD152+ T regulatory cells were also significantly inhibited by IM in a dose-related fashion. These findings suggest that the clinical impact IM must not necessarily result in a reduced efficacy of the graft-versus-leukemia effect observed after allo-SCT and/or DLIs but might depend rather on the ratio of CD8+ cytotoxic T cells and CD4+CD25hi T regulatory cells.

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Flow cytometric analysis of T lymphocytes and cytokines in aqueous humor of patients with varicella zoster virus-mediated acute retinal necrosis

BMC Ophthalmology ◽

10.1186/s12886-021-01951-1 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Hao Kang ◽

Yunbo Wei ◽

Ming Liu ◽

Di Yu ◽

Yong Tao

Keyword(s):

T Lymphocytes ◽

Varicella Zoster Virus ◽

Aqueous Humor ◽

T Lymphocyte ◽

Lymphocyte Subsets ◽

Flow Cytometric Analysis ◽

T Lymphocyte Subsets ◽

Varicella Zoster ◽

Flow Cytometric ◽

Cd8 T Lymphocytes

Abstract Background The purpose of this study is to investigate the aqueous humor (AH) T lymphocyte subsets and cytokines of acute retinal necrosis (ARN) to elucidate the immunologic inflammatory features of this disorder. Methods Three patients with ARN infected with varicella zoster virus (VZV) who underwent multiple intravitreal injections of ganciclovir were enrolled in this study. The control group consisted of four non-infectious patients with acute anterior uveitis (AAU). Flow cytometric analysis was performed on the lymphocyte subsets from the AH and peripheral blood (PB) samples during the active phase of intraocular inflammation. Five inflammatory cytokines were measured in each AH sample and various clinical characteristics were also assessed. Results VZV deoxyribonucleic acid (DNA) was detected by real-time polymerase chain reaction (PCR) in AH from all the ARN patients, who showed higher CD8+ T lymphocytes population in AH than the AAU patients (p = 0.006). CD4/CD8 ratios of T lymphocytes and the percentage of CD8 + CD25+ T lymphocytes in AH were significantly lower in ARN than in AAU (p = 0.006; p = 0.012). In the ARN patients, the percentages of CD4+ and CD8+ T lymphocytes in AH were higher than those found in PB. The percentage of CD4 + CD25+ T lymphocytes in AH was significantly higher than the proportion in PB in the AAU patients (p = 0.001). Immunoregulatory cytokine Interleukin-10 in AH was significantly elevated in the ARN patients in comparison with the case of the AAU patients (p = 0.036). In ARN, the copy number of VZV DNA in AH positively correlated with the percentage of CD8+ T lymphocytes in AH and negatively correlated with the CD4/CD8 ratio in AH during the course of disease treatment (p = 0.009, r = 0.92; p = 0.039, r = − 0.834). Conclusion The ARN patients caused by VZV had different intraocular T lymphocyte subsets and cytokines profile than those of the non-infectious patients. High percentages of CD8+ T lymphocytes and low CD4/CD8 T cell ratios may be a potential biomarker for diagnosis of viral-infectious uveitis. T lymphocytes examination at the inflammatory sites has the potential to become a useful research tool for differentiating viral and non-viral uveitis.

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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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Flow Cytometric Analysis of T Lymphocytes and Cytokines in Aqueous Humor of Patients with Varicella Zoster Virus-mediated Acute Retinal Necrosis

10.21203/rs.3.rs-56692/v1 ◽

2020 ◽

Author(s):

Hao Kang ◽

Yunbo Wei ◽

Ming Liu ◽

Di Yu ◽

Yong Tao

Keyword(s):

T Lymphocytes ◽

Varicella Zoster Virus ◽

Aqueous Humor ◽

T Lymphocyte ◽

Lymphocyte Subsets ◽

Flow Cytometric Analysis ◽

T Lymphocyte Subsets ◽

Varicella Zoster ◽

Flow Cytometric ◽

Cd8 T Lymphocytes

Abstract Background: The purpose of this study is to investigate the aqueous humor (AH) T lymphocyte subsets and cytokines of acute retinal necrosis (ARN) to elucidate the immunologic inflammatory features of this disorder.Methods: Three patients with ARN infected with varicella zoster virus (VZV) who underwent multiple intravitreal injections of ganciclovir were enrolled in this study. The control group consisted of four non-viral infectious patients with acute anterior uveitis (AAU). Flow cytometric analysis was performed on the lymphocyte subsets from the AH and peripheral blood (PB) samples during the active phase of intraocular inflammation. Five inflammatory cytokines were measured in each AH sample and various clinical characteristics were also assessed.Results: VZV DNA was detected by real-time polymerase chain reaction (PCR) in AH from all the ARN patients, who showed higher CD8+ T lymphocytes population in AH than the AAU patients (p=0.006). CD4/CD8 ratios of T lymphocytes and the percentage of CD8+CD25+ T lymphocytes in AH were significantly lower in ARN than in AAU (p=0.006; p=0.012). In the ARN patients, the percentages of CD4+ and CD8+ T lymphocytes in AH were higher than those found in PB. The percentage of CD4+CD25+ T lymphocytes in AH was significantly higher than the proportion in PB in the AAU patients (p=0.001). Immunoregulatory cytokine Interleukin-10 in AH was significantly elevated in the ARN patients in comparison with the case of the AAU patients (p=0.036). In ARN, the copy number of VZV DNA in AH positively correlated with the percentage of CD8+ T lymphocytes in AH and negatively correlated with the CD4/CD8 ratio in AH during the course of disease treatment (p=0.009, r=0.92; p=0.039, r=-0.834).Conclusion: The ARN patients caused by VZV had different intraocular T lymphocyte subsets and cytokines profile than those of the non-viral infectious patients. High percentages of CD8+ T lymphocytes and low CD4/CD8 T cell ratios may be a potential biomarker for diagnosis of viral-infectious uveitis. T lymphocytes examination at the inflammatory sites has the potential to become a useful research tool for differentiating viral and non-viral uveitis.

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The Effects of Mesenchymal Stem Cells on Antimelanoma Immunity Depend on the Timing of Their Administration

Stem Cells International ◽

10.1155/2020/8842659 ◽

2020 ◽

Vol 2020 ◽

pp. 1-13

Author(s):

Dragana Miloradovic ◽

Dragica Miloradovic ◽

Bojana Simovic Markovic ◽

Aleksandar Acovic ◽

Carl Randall Harrell ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

T Lymphocytes ◽

Tumor Growth ◽

Nk Cells ◽

Plasma Levels ◽

Th17 Cells ◽

Antitumor Immunity ◽

Granzyme B ◽

Lower Plasma

There is still a lively debate about whether mesenchymal stem cells (MSCs) promote or suppress antitumor immune response. Although several possible explanations have been proposed, including different numbers of injected and engrafted MSCs, heterogeneity in phenotype, and function of tumor cells, the exact molecular mechanisms responsible for opposite effects of MSCs in modulation of antitumor immunity are still unknown. Herewith, we used a B16F10 murine melanoma model to investigate whether timing of MSC administration in tumor-bearing mice was crucially important for their effects on antitumor immunity. MSCs, intravenously injected 24 h after melanoma induction (B16F10+MSC1d-treated mice), significantly enhanced natural killer (NK) and T cell-driven antitumor immunity, suppressed tumor growth, and improved survival of melanoma-bearing animals. Significantly higher plasma levels of antitumorigenic cytokines (TNF-α and IFN-γ), remarkably lower plasma levels of immunosuppressive cytokines (TGF-β and IL-10), and a significantly higher number of tumor-infiltrating, IFN-γ-producing, FasL- and granzyme B-expressing NK cells, IL-17-producing CD4+Th17 cells, IFN-γ- and TNF-α-producing CD4+Th1 cells, and CD8+cytotoxic T lymphocytes (CTLs) were observed in B16F10+MSC1d-treated mice. On the contrary, MSCs, injected 14 days after melanoma induction (B16F10+MSC14d-treated mice), promoted tumor growth by suppressing antigen-presenting properties of tumor-infiltrating dendritic cells (DCs) and macrophages and by reducing tumoricidal capacity of NK cells and T lymphocytes. Significantly higher plasma levels of TGF-β and IL-10, remarkably lower plasma levels of TNF-α and IFN-γ, and significantly reduced number of tumor-infiltrating, I-A-expressing, and IL-12-producing macrophages, CD80- and I-A-expressing DCs, granzyme B-expressing CTLs and NK cells, IFN-γ- and IL-17-producing CTLs, CD4+Th1, and Th17 cells were observed in B16F10+MSC14d-treated animals. In summing up, the timing of MSC administration into the tumor microenvironment was crucially important for MSC-dependent modulation of antimelanoma immunity. MSCs transplanted during the initial phase of melanoma growth exerted tumor-suppressive effect, while MSCs injected during the progressive stage of melanoma development suppressed antitumor immunity and enhanced tumor expansion.

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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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