Increased Cytotoxic T Cells and NK Cells in Patients With Myeloma by Flow Cytometry

ABSTRACTAlthough PD-1 was shown to be a hallmark of T cells exhaustion, controversial studies have been reported on the role of PD-1 on NK cells. Here, we found by flow cytometry and single cell RNA sequencing analysis that PD-1 can be expressed on MHC class I-deficient tumor-infiltrating NK cells in vivo. We also demonstrate distinct alterations in the phenotype of PD-1-deficient NK cells which in part could be attributed to a decrease in tumor-infiltrating NK cells in PD-1-deficient mice. NK cells from PD-1-deficient mice exhibited a more mature phenotype which might reduce their capacity to migrate and kill in vivo. Finally, our results demonstrate that PD-L1 molecules in membranes of PD-1-deficient NK cells migrate faster than in NK cells from wildtype mice, suggesting that PD-1 and PD-L1 form cis interactions with each other on NK cells.

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Cellular investigations

Oxford Handbook of Clinical Immunology and Allergy ◽

10.1093/med/9780199603244.003.0020 ◽

2013 ◽

pp. 555-578

Author(s):

Gavin P Spickett

Keyword(s):

Flow Cytometry ◽

Tissue Culture ◽

T Cells ◽

Bronchoalveolar Lavage ◽

Cytotoxic T Cells ◽

Cd40 Ligand ◽

Regulatory Factors ◽

Ligand Expression ◽

Proliferation Assays ◽

Protein Assays

Introduction Flow cytometry Tissue culture Proliferation assays Immunohistology Cytokine, chemokine, soluble protein assays Apoptosis assays Adhesion markers Bronchoalveolar lavage (BAL) studies CD40 ligand expression Complement membrane regulatory factors Cytokine and cytokine receptor measurement Cytotoxic T cells FOXP3 (regulatory T cells—IPEX syndrome) Genetic and protein studies...

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Sinonasal T-Cell Expression of Cytotoxic Mediators Granzyme B and Perforin is Reduced in Patients with Chronic Rhinosinusitis

American Journal of Rhinology and Allergy ◽

10.2500/ajra.2017.31.4474 ◽

2017 ◽

Vol 31 (6) ◽

pp. 352-356 ◽

Cited By ~ 1

Author(s):

Sarah E. Smith ◽

Rodney J. Schlosser ◽

James R. Yawn ◽

Jose L. Mattos ◽

Zachary M. Soler ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

T Cell ◽

Nk Cells ◽

Chronic Rhinosinusitis ◽

Nasal Polyp ◽

Granzyme B ◽

Cytotoxic Cells ◽

Sinonasal Mucosa ◽

Perforin Expression

Background CD8+ T cells and natural killer (NK) cells are cytotoxic cells that use granzyme B (GrB) and perforin. Defective cytotoxic function is known to play a role in dysregulated immune response as seen in chronic sinusitis, also referred to as chronic rhinosinusitis (CRS). However, to our knowledge, in the United States, neither GrB or perforin expression has been reported in patients with CRS. Objective The aim of this study was to investigate sinonasal cytotoxic cells, their mediators, and cell-specific distribution of these mediators in patients with CRS with nasal polyp (CRSwNP) and in patients with CRS without nasal polyp (CRSsNP). Methods Blood and sinus tissue samples were taken from patients with CRSsNP (n = 8) and CRSwNP (n = 8) at the time of surgery. Control subjects (n = 8) underwent surgery for cerebrospinal fluid leak repair or to remove non-hormone-secreting pituitary tumors. The cells were analyzed via flow cytometry by using CD8 expression to identify cytotoxic T cells and CD56 expression to identify NK cells. Intracellular GrB and perforin expression were analyzed with flow cytometry. Results We observed no significant differences in plasma or peripheral blood immune cell numbers or specific levels of GrB or perforin among the groups. In the sinonasal mucosa of the patients with CRSsNP and the patients with CRSwNP, there was a significant decrease in GrB and perforin levels (p <0.05) despite similar or increased numbers of cytotoxic cells when compared with the controls. The overall decrease in GrB and perforin in the sinonasal mucosa of the patients with CRSsNP and the patients with CRSwNP was due to decreased T cell production. There was no difference in total NK cell count or expression of perforin or GrB among all the groups. Conclusion Total levels of sinonasal GrB and perforin were decreased in the sinonasal mucosa of both the patients with CRSwNP and the patients with CRSsNP compared with the controls, whereas sinonasal CD8+ T cells, (but not NK cells,), intracellular stores of GrB and perforin were reduced in the patients with CRSwNP compared with the controls.

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Platelet-derived growth factor mediates survival of leukemic large granular lymphocytes via an autocrine regulatory pathway

Blood ◽

10.1182/blood-2009-06-223719 ◽

2010 ◽

Vol 115 (1) ◽

pp. 51-60 ◽

Cited By ~ 49

Author(s):

Jun Yang ◽

Xin Liu ◽

Susan B. Nyland ◽

Ranran Zhang ◽

Lindsay K. Ryland ◽

...

Keyword(s):

T Cells ◽

Growth Factor ◽

Nk Cells ◽

Nk Cell ◽

Cytotoxic T Cells ◽

Platelet Derived Growth Factor ◽

Leukemic Cells ◽

Immunocytochemical Staining ◽

Term Survival ◽

Lgl Leukemia

Abstract Large granular lymphocyte (LGL) leukemia results from chronic expansion of cytotoxic T cells or natural killer (NK) cells. Apoptotic resistance resulting from constitutive activation of survival signaling pathways is a fundamental pathogenic mechanism. Recent network modeling analyses identified platelet-derived growth factor (PDGF) as a key master switch in controlling these survival pathways in T-cell LGL leukemia. Here we show that an autocrine PDGF regulatory loop mediates survival of leukemic LGLs of both T- and NK-cell origin. We found high levels of circulating PDGF-BB in platelet-poor plasma samples from LGL leukemia patients. Production of PDGF-BB by leukemic LGLs was demonstrated by immunocytochemical staining. Leukemic cells expressed much higher levels of PDGFR-β transcripts than purified normal CD8+ T cells or NK cells. We observed that phosphatidylinositol-3-kinase (PI3 kinase), Src family kinase (SFK), and downstream protein kinase B (PKB)/AKT pathways were constitutively activated in both T- and NK-LGL leukemia. Pharmacologic blockade of these pathways led to apoptosis of leukemic LGLs. Neutralizing antibody to PDGF-BB inhibited PKB/AKT phosphorylation induced by LGL leukemia sera. These results suggest that targeting of PDGF-BB, a pivotal regulator for the long-term survival of leukemic LGLs, may be an important therapeutic strategy.

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Development and Function of NK and T Cells in AML Patients after Allogeneic Stem Cell Transplantation (SCT).

Blood ◽

10.1182/blood.v104.11.3244.3244 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3244-3244

Author(s):

Gabriele Multhoff ◽

Catharina Gross ◽

Anne Dickinson ◽

Ernst Holler

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

T Cells ◽

Stem Cell ◽

Nk Cells ◽

Nk Cell ◽

Granzyme B ◽

Hla Class I ◽

K562 Cells ◽

Cytolytic Response

Abstract Purpose: Hsp70 was frequently found on the plasma membrane of bone marrow-derived leukemic blasts, but not on normal bone marrow cells. Hsp70 membrane expression could be correlated with protection against therapy-induced apoptosis (Nylandsted et al 2004). In contrast, these tumor cells have been found to be highly sensitive to the cytolytic attack mediated by NK cells. In vitro, Hsp70-activated NK cells efficiently lysed autologous Hsp70 membrane-positive leukemic blasts (Gehrmann et al 2003). Granzyme B release served as a surrogate marker for estimating the cytolytic response of NK cells against Hsp70 membrane-positive tumor target cells (Gross et al 2003). Here, we studied the development of NK and T cells in AML patients (n=6) after allogeneic SCT at different time points (days 14–20, 45, 90, 180, 1 year) after allogeneic stem cell transplantation (SCT). Methods: HLA class I, HLA-E and Hsp70 surface expression was determined on all patient-derived leukemic blasts of the bone marrow by flow cytometry. The amount of NK and T cells was investigated by multicolor flow cytometry using CD3/ CD16 and CD56 and CD94/ CD56 antibody-combinations detecting NK cell specific markers. Effector cell function was tested in a granzyme B ELISPOT assay against patient-derived leukemic blasts and K562 cells. Results: All tested leukemic blasts were positive for HLA class I, HLA-E, and Hsp70. After induction therapy the amount of CD3-negative, CD56/CD94-positive NK cells was 28±16%, that of CD3-positive T cells was 58±3%. On days 14–21 after allogeneic SCT, 58±9% of the donor-derived peripheral blood lymphocytes (PBL) were CD3-negative, CD56/CD94-positive NK cells; the amount of CD3-positive T cells was 26±7.5%. On day 45, the amount of NK cells further increased up to 68±7.9%; that of T cells further decreased down to 16±5.6%. On day 90 and day 180 the amount of NK cells was still 41±10%; that of T cells was 29±12%. Interestingly, high NK cell counts correlated with an increased cytolytic response against leukemic blast and K562 cells. One year after allogeneic SCT, NK (20±1%) and T cell (52±18%) ratios were comparable to that of healthy human individuals. Conclusions: Between days 14 and 180 after allogeneic SCT, the amount of NK cells was significantly elevated if compared to that of T cells. Concomitantly, cytolytic function against leukemic blasts was significantly elevated. Normal levels, in the composition of NK and T cells were reached 1 year after SCT. Project funded by EU-TRANS-EUROPE grant QLK3-CT-2002-01936.

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KIR Positive Subsets of Human CD56+CD3+ NKT Cells Are Different in Frequency from Equivalant KIR Positive CD56+CD3- NK Cell Subsets.

Blood ◽

10.1182/blood.v110.11.3878.3878 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3878-3878

Author(s):

Ilka Bondzio ◽

Andreas Arendt ◽

Jurgen Schmitz ◽

Volker Huppert

Keyword(s):

T Cells ◽

Nk Cells ◽

Nk Cell ◽

Cytotoxic T Cells ◽

Killer Cell ◽

Nkt Cells ◽

Nkt Cell ◽

Cell Clones ◽

Number Of Publications

Abstract Killer cell immunoglobulin-like receptors (KIRs) are known to modulate the cytotoxic ability of human Natural Killer (NK) cells, as well as a subset of T cells. To date, only a very small number of publications have discussed the role of KIRs on T cells, e.g. CMV-specific CD4+CD28-KIR+ cytotoxic T cells (van Bergen, J., J Immunol. 2004), so we investigated whether CD56+CD3+ NKT cells might also have KIR-positive subsets. Whole human blood as well as magnetically sorted human CD56+CD3+ NKT cells were analyzed for their expression of various KIR molecules using a novel panel of fluorochrome-conjugated, anti-KIR monoclonal antibodies (CD158a/h (KIR2DL1/DS1), CD158b (KIR2DL2), CD158e (KIR3DL1), CD158i (KIR2DS4), KIR2D; Miltenyi Biotec). KIR-positive CD56+CD3+ NKT cells were identified in every donor tested. Donors possessing NK cells of a specific KIR phenotype also possessed CD56+CD3+ NKT cells with the same KIR phenotype. KIRs were also expressed in a clonal fashion on CD56+CD3+ NKT cells, similarly to NK cells. The investigated KIRs were also shown to be expressed on unseparated NK and CD56+CD3+ NKT cells from whole blood. In addition, the ratio between KIR expression on NK and CD56+CD3+ NKT cells was calculated for each donor analyzed. The results show that there is no correlation between the frequencies of KIR expression on NK cells with that of CD56+CD3+ NKT cells. For example, the expression of CD158a/h in one donor was found to be the highest of all CD56+CD3+ NKT cells analyzed, but the lowest of all NK cells by comparison to the other donors tested. For all KIR phenotypes analyzed, the frequency of KIR+ NK cells was higher than the frequency of KIR+ CD56+CD3+ NKT cells in all samples (range: 1.1 to 25.3-fold higher). Interestingly, the frequency of KIR+ NK cells versus KIR+ CD56+CD3+ NKT cells differs significantly between donors: in one donor the frequency of KIR expression is between 7.3 to 25.3-fold higher in NK cells for multiple KIR phenotypes, while this range is more narrow in other donors (2.0–5.4-fold higher). The frequencies of CD56+CD3+ NKT cell subsets staining positive for particular KIRs differ significantly between donors, e.g. for CD158b, the number of positive CD56+CD3+ NKT cells fall within a range of 4.8% to 43.3%. For CD56+CD3+ NKT cells sorted with MACS® Technology, a similarly wide-ranging distribution of CD158b (KIR2DL2) expression was found (0.85%–5.82%), though at a lower level. Further research will be required to explore these differences as they may point to different mechanisms of KIR regulation. The identification of KIR-positive CD56+CD3+ NKT cells may also provide an opportunity for their use for functional KIR studies instead of NK cell clones, as the cloning of CD56+CD3+ NKT cells may prove easier (i.e. using standard T cell cloning methods) than that of NK cells.

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Trephine biopsies are enriched for activated T/NK cells and cytotoxic T cells

Immunology Letters ◽

10.1016/j.imlet.2005.01.008 ◽

2005 ◽

Vol 99 (1) ◽

pp. 94-102 ◽

Cited By ~ 4

Author(s):

J DEAN ◽

D MCCARTHY ◽

L GOLDENMASON ◽

C OFARRELLY

Keyword(s):

T Cells ◽

Nk Cells ◽

Cytotoxic T Cells

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DETECTION OF CYTOMEGALOVIRUS-SPECIFIC CYTOTOXIC T-CELLS IN TRANSPLANT RECIPIENTS USING THE CYTOKINE FLOW CYTOMETRY ASSAY.

Journal of Investigative Medicine ◽

10.1097/00042871-200401001-00407 ◽

2004 ◽

Vol 52 ◽

pp. S150

Author(s):

R. Rhada ◽

M. Toyoda ◽

A. Pao ◽

D. Puliyanda ◽

S. C. Jordan

Keyword(s):

Flow Cytometry ◽

T Cells ◽

Cytotoxic T Cells ◽

Transplant Recipients ◽

Flow Cytometry Assay ◽

Cytokine Flow Cytometry

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Pharmacokinetic and Pharmacodynamic Study of NKTR-255, a Polymer-Conjugated Human IL-15, in Cynomolgus Monkey

Blood ◽

10.1182/blood-2018-99-115638 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2952-2952

Author(s):

Takahiro Miyazaki ◽

Peiwen Kuo ◽

Mekhala Maiti ◽

Palakshi Obalapur ◽

Murali Addepalli ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

Nk Cells ◽

Cd8 T Cells ◽

Cynomolgus Monkey ◽

Memory T Cells ◽

Granzyme B ◽

Employment Equity ◽

Equity Ownership ◽

Dose Dependent

Abstract Introduction IL-15 is a common gamma chain cytokine that activates and provides a survival benefit to T-cells and NK cells and has long been recognized as having potential as an immunotherapeutic agent for the treatment of cancer. Therapeutic use of native IL-15 has been challenging due to, for example, its unfavorable pharmacokinetic and safety properties. NKTR-255 is a polymer-conjugated human IL-15 that retains binding affinity to the alpha subunit of IL-15 receptor and exhibits reduced clearance to thereby provide a sustained pharmacodynamics response. Here we investigate the biological effects of NKTR-255 in naïve cynomolgus monkey. Methods In vitro monkey whole blood was treated with NKTR255 and the percentage of pSTAT5 positive populations in each NK, CD4 T and CD8 T cells was determined by flow cytometry. In an PK/PD study, monkeys received single IV doses of 0.001, 0.003, 0.01, 0.03, or 0.1 mg/kg NKTR-255. Blood samples were collected to determine the plasma concentrations of NKTR-255 and to assess the effects of NKTR-255 on NK and CD8 T cells at multiple time points; flow cytometry was used to measure STAT5 phosphorylation, Ki-67 expression and frequency of cell populations. Granzyme B expression was assessed in NK and CD8 T cells by flow cytometry. Results NKTR-255 induced dose-dependent phosphorylation of STAT5 in monkey whole blood (EC50 values NK cells: 6.9 ng/ml, CD8 T cells: 39 ng/ml, CD4 T cells: 53 ng/ml). The half-life and clearance of NKTR-255 were 26x longer and 38x lower, respectively, than IL-15. NKTR-255 engaged the IL-15 signaling pathway, in vivo, demonstrating both robust and sustained STAT5 phosphorylation in lymphocytes. NKTR-255 drove the proliferation of total CD8 T cells and NK cells in a dose-dependent manner, with dramatic and durable increases observed in Ki67 positive population and absolute cell numbers (NK cells: 6.1 fold; CD8 T cells: 7.8 fold from baseline on day 5 at 0.1 mg/kg). These effects were strongly biased towards CD8 T cells and NK cells, with substantially less induction of CD4 T cells. The Ki67 response analyses of the T cell subpopulation revealed a higher response of memory populations than for naive T cells. Among memory T cells, effector memory T cells showed the highest response over stem cell memory T cells and central memory T cells. Finally, NKTR-255 also increased the expression of Granzyme B in both NK and CD8 T cells, concomitant with an enhancement in target cell lysis. Conclusions Nektar has generated a novel and potent molecule in NKTR-255 that not only preserves the relevant biology of IL-15, but additionally provides enhanced PK and PD properties relative to the native IL-15 cytokine. NKTR-255 is being developed as an immune-stimulatory agent to target NK and CD8 T cell biology for the treatment of cancer. Disclosures Miyazaki: Nektar Therapeutics: Employment, Equity Ownership. Kuo:Nektar Therapeutics: Employment, Equity Ownership. Maiti:Nektar Therapeutics: Employment, Equity Ownership. Obalapur:Nektar Therapeutics: Employment, Equity Ownership. Addepalli:Nektar Therapeutics: Employment, Equity Ownership. Rubas:Nektar Therapeutics: Employment, Equity Ownership. Sims:Nektar Therapeutics: Employment, Equity Ownership. Zhang:Nektar Therapeutics: Employment, Equity Ownership. Madakamutil:Nektar Therapeutics: Employment, Equity Ownership. Zalevsky:Nektar Therapeutics: Employment, Equity Ownership.

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