Chronic Myeloid Leukemia Patients Undergoing Interferon Alpha Therapy Exhibit Normal Peripheral Blood Gamma Delta T Cells That May Be Expanded in Vitro to Generate Predominantly CD45RA-Positive Effector Memory Cells for Immunotherapy

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3729-3729
Author(s):  
Gabrielle Melanie Siegers ◽  
Anna Kreutzman ◽  
Mette Matilda Ilander ◽  
Satu Mustjoki ◽  
Lynne Marie Postovit
Keyword(s):  
T Cells ◽  
Chronic Myeloid Leukemia ◽  
Peripheral Blood ◽  
Myeloid Leukemia ◽  
Healthy Donor ◽  
Effector Memory ◽  
Gamma Delta T Cells ◽  
Healthy Controls ◽  
Gamma Delta

Abstract Abstract 3729 Comprising <10% of T lymphocytes in human blood, gamma delta T cells (GDTc) are cytotoxic immune surveillance cells that are an attractive option for adoptive immunotherapy. We have previously shown that healthy donor GDTc expanded in vitro are cytotoxic to EM-2 and K562 Ph+ leukemia cell lines but not to autologous peripheral blood mononuclear cells (PBMCs). Additionally, we have noted an oligoclonal expansion of GDTc in interferon alpha (IFNα) monotherapy treated chronic myeloid leukemia (CML) patients. The current study is aimed at elucidating the role that these cells play in the remission of IFNα-treated CML patients. Immunophenotyping of thawed PBMC samples from CML patients undergoing (IFN-ON) or who had discontinued IFNα monotherapy (IFN-OFF) revealed similar central CD45RA-CD27+ (5.7% ± 2.7%, mean ± standard error, n=6) and CD45RA-CD27- effector memory (3.7% ± 2.0%, n=6) GDTc pools compared to those from healthy age-matched donors (4.4% ± 1.2% and 4.7% ± 1.4% respectively, n=3), suggesting that GDTc pools are normal in these patients. However, differences approaching significance were observed between IFN-ON (n=3) and healthy controls (n=3), with IFN-ON exhibiting less CD45RA+CD27+ naïve GDTc (24.6% ± 6.7% compared to 48.2% ± 7.7%, p=0.08) and more terminally differentiated CD45RA+CD27- RA+ effector memory GDTc (66.4% ± 11.8% compared to 42.7 ± 7.3%, p=0.16). The percentage of GDTc expressing the activating Natural Killer cell receptor NKG2D was more variable and significantly lower in freshly thawed IFN-OFF patient samples (53.1% ± 4.6%, p=0.03) as compared to age-matched healthy donor controls (68.4% ± 1.5%). A similar trend was also observed between IFN-ON patients and healthy controls (IFN-ON 54.5% ± 8.8%, p=0.08). Although GDTc are typically difficult to recover and expand from cryopreserved PBMCs, we have developed a new protocol with which we can expand patient GDTc, potentially enabling the generation of therapeutic doses on demand. We achieved variable expansions of 41- to 9097-fold from patient samples over 21 days, with four of six patient cultures achieving over 100-fold GDTc expansion. Of these four cultures, two were from each patient group, suggesting that IFN-ON or –OFF status did not correlate with the ability of these cells to expand in vitro. Importantly, expanded GDTc were mostly terminally differentiated CD45RA+CD27- RA+ effector memory GDTc (52% ± 11.7%, n=6). Of the total GDTc population, 65.9% ± 4.6% expressed NKG2D (n=6), suggesting that our protocol can generate terminally differentiated cytotoxic GDTc. We are now investigating whether the cells expanded in vitro are the clonal GDTc population previously identified in our patients and whether these expanded cells are able to kill Ph+ leukemia cells. Our findings will be important not only for understanding the mechanism of IFNα-induced cure of the patients, but also for developing GDTc therapies for CML and other malignancies. Disclosures: Mustjoki: Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria.

Dendritic Cells Can Be Primed to Concurrently Activate Both Unconventional Gamma/delta and Conventional alpha/beta T Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3893-3893
Author(s):  
Francesca Fiore ◽  
Barbara Castella ◽  
Barbara Nuschak ◽  
Raffaello Bertieri ◽  
Sara Mariani ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
Immune Responses ◽  
Tumor Cells ◽  
Peripheral Blood ◽  
Adoptive Cell Therapy ◽  
Effector Memory ◽  
Gamma Delta T Cells ◽  
Gamma Delta ◽  
Alpha Beta

Abstract Vgamma9/Vdelta2 (gamma/delta) T cells represent the major subset of unconventional T cells circulating in the peripheral blood. Gamma/delta T cells play a major role in immune defenses against microbes, stressed cells and tumor cells. This property is based on their capability to naturally recognize phosphoantigens (pAgs), which are produced via the mevalonate (Mev) or the DOXP pathway in mammalian and nonmammalian cells, and induced self-ligands, which are de novo expressed or upregulated on the surface of stressed or tumor cells. Interestingly, gamma/delta T cells can also be activated by aminobisphosphonates (ABP)-treated monocytes. We have previously shown that ABP specifically target the Mev pathway of monocytes and induce the accumulation of phosphorylated Mev metabolites naturally recognized by gamma/delta T cells. The aim of this work was to determine whether ABP-treated dendritic cells (DC) can also activate gamma/delta T cells and whether this activation, if any, is detrimental or beneficial to the generation of antigen (Ag)-specific MHC-restricted immune responses mediated by conventional alpha/beta T cells. To this end, we have generated highly purified immature (iDC) and mature DC (mDC) from peripheral blood monocytes of healthy donors and incubated with zoledronic acid (Zol) for 24 hours. Zol is the most potent ABP currently available for clinical use. Zol treatment did not affect the phenotype and immunostimulatory properties of iDC and mDC. Zol-treated iDC and mDC induced a rapid and vigorous expansion of central memory and effector memory gamma/delta T cells. Zol-treated iDC were more potent inducers of gamma/delta T-cell activation than mDC and monocytes. Activated gamma/delta T cells displayed antitumor activity and expressed on the cell surface the appropriate antigen repertoire to target secondary lymphoid organs and exert costimulatory activity on conventional alpha/beta T cells. Indeed, an in vitro model showed that antigen-specific MHC-restricted immune responses againt the influenza matrix peptide were significantly improved by the concurrent activation of gamma/delta T cells. This is the first report showing that: 1) DC can simultaneously be primed to activate both gamma/delta and alpha/beta T cells; 2) the former act as cellular adjuvants for the development of adaptive immune responses. In conclusion, large numbers of gamma/delta T cells with effector and costimulatory activities can rapidly be generated by Zol-treated iDC/mDC. This strategy is worth of further investigation to improve adoptive cell therapy and vaccine interventions against tumors and infections.

PD-1 (CD279) Contributes to the Exhaustion of Gamma/Delta-T Cells in Tumor-Bearing Mice

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 839-839 ◽  
Author(s):  
Richard D Lopez ◽  
Shin Mineishi ◽  
Lawrence S. Lamb ◽  
Hyung-Gyoon Kim ◽  
Benjamin Beck
Keyword(s):  
T Cells ◽  
Peripheral Blood ◽  
Ex Vivo ◽  
Gamma Delta T Cells ◽  
Gamma Delta ◽  
Tumor Bearing ◽  
Functionally Impaired ◽  
Alpha Beta

Abstract Abstract 839 Objectives: Programmed death-1 (PD-1)/CD279 is an immunoinhibitory receptor that can be physiologically expressed on activated antigen-specific alpha/beta T-cells and is thought to play a role in maintaining a balance between T-cell activation and tolerance. Recently, both in vitro and in vivo, it has been shown that disrupting the interaction between PD-1 and its ligands can improve antitumor effects in preclinical and clinical models, this suggesting an important role played by this pathway in escape from immune surveillance. In comparison to healthy donors, gamma/delta-T cells found in tumor-bearing hosts can be diminished in number, or can be functionally impaired in a variety of important ways. While the mechanisms accounting for these numeric or functional defects have remained unclear, here we examine the extent to which PD-1 expression on gamma/delta-T cells may play a role in this process. Findings: We first noted that peripheral blood gamma/delta-T cells are diminished in numbers in patients newly diagnosed with cancer. In addition, these gamma/delta-T cells expanded poorly when cultured ex vivo. Similar to humans, in tumor-bearing mice, we found that peripheral blood gamma/delta-T cells are diminished in number and likewise, expand poorly when cultured ex vivo. Using FACS analysis of mouse peripheral blood, we first determined that a substantial proportion of gamma/delta-T cells are actively undergoing apoptosis in tumor-bearing mice compared to healthy mice. Further analysis revealed that PD-1 is significantly upregulated on gamma/delta-T cells taken from tumor-bearing mice compared to gamma/delta-T cells taken from healthy mice. In contrast, no difference of PD-1 expression was seen when comparing alpha/beta-T cells taken from tumor-bearing and healthy mice. Using in vitro co-culture studies, we next determined that apoptosis in gamma/delta-T cells can be induced by direct contact with malignant cells, but not by contact with non-malignant cells. We then showed in these cultures that PD-1 is upregulated on gamma/delta-T cells co-cultured with tumor cell lines. Moreover, we were able to determine that the PD-1-positive gamma/delta-T cells in these cultures were undergoing apoptosis to a greater extent than PD-1-negative gamma/delta-T cells in these same cultures. Finally, in vitro using CFSE-based methods, we showed that while gamma/delta-T cells isolated from healthy mice readily proliferate upon mitogen stimulation, in contrast, gamma/delta-T cells from tumor-bearing mice proliferate poorly under the same conditions. However, in spleen cell cultures derived from tumor-bearing mice, upon addition of a monoclonal antibody directed against PD-L1 (B7-H1), a ligand for PD-1, substantial restoration of gamma/delta-T cell proliferation occurs. Conclusion: Until now, the role played by PD-1 in the exhaustion of tumor-reactive gamma/delta T-cells has not been explored. Using in vitro and in vivo models, we show that the PD-1 pathway is a potentially important mechanism by which gamma/delta T-cells are either functionally impaired or otherwise exhausted in tumor-bearing mice. These findings suggest that by disrupting the PD-1 pathway, it may be possible to “revive” or “rescue” gamma/delta T-cells in tumor-bearing hosts. Disclosures: No relevant conflicts of interest to declare.

600 In vitro anticancer and immunomodulatory activities of NBT-167, a dimer of resveratrol

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A635-A635
Author(s):  
Jeffrey Zhang ◽  
Everett Henry ◽  
L Harris Zhang ◽  
Wanying Zhang
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cancer Cells ◽  
Cancer Cell ◽  
Immune Cells ◽  
Cell Killing ◽  
Gamma Delta T Cells ◽  
Gamma Delta ◽  
Raw264.7 Macrophages

BackgroundResveratrol (3,4’,5-trihydroxystilbene), a stilbenoid isolated from many species of plants, is widely known for its antioxidative, anti-inflammatory, immunomodulatory and anticancer activities. Recently, novel resveratrol oligomers have been isolated from various plants; their diverse structures are characterized by the polymerization of two or more resveratrol units. Little is known regarding the anticancer and immunomodulating activities of these oligomers. In this study, we designed in vitro models to compare resveratrol side by side with its natural dimer NBT-167 for their anticancer and immunological activities.MethodsWe isolated resveratrol and its dimer (NBT-167) from plants. The potency of the compounds was compared side by side using cancer cell survival assays and immunological assays with various types of human cells including cancer cell lines, PBMCs and enriched NK, gamma delta T cells, THP-1 monocytic cells, HL-60 promyelocytic leukemia cells as well as mouse RAW264.7 macrophages.ResultsNBT-167 was found to be more potent than resveratrol in inhibiting growth of various cancer cells and modulation of cytokine production from anti-IgM, LPS, PHA or SEB stimulated PBMC. Both compounds similarly enhanced IL-2 stimulated NK and gamma delta T cell killing activity against K562 cells and modulated nitric oxide production from LPS/IFN-g induced RAW264.7 macrophages and phagocytotic activity of HL-60 cells. NBT-167 was slightly more potently than resveratrol in inhibiting chemotaxis of HL-60 cells and blocking cell cycle of THP-1 and HL-60 cells at G1/S transition. In addition, NBT-167, but not resveratrol, could increase IL-2 production and T cell proliferation stimulated with anti-CD3 and anti-CD28 and synergize with anti-PD-1 antibody to increase IL-2 and IFN-gamma production in co-culture of allotypic T cells and dendric cells (MLR).ConclusionsOur data showed that NBT-167, a dimer of resveratrol, had anticancer and immunomodulatory activities such as modulation of expression of cytokines in immune cells and induction of cancer cell-killing activities of NK and gamma delta T cells. Generally, NBT-167 appeared to have higher activities than resveratrol in modulating immune cells and inhibiting cancer cells. NBT-167 could be a promising cancer immunotherapeutic agent targeting both cancer cells and immune cells.

scholarly journals Transient increase in circulating gamma/delta T cells during Plasmodium vivax malarial paroxysms.

1994 ◽  
Vol 179 (1) ◽  
pp. 311-315 ◽  
Author(s):  
M K Perera ◽  
R Carter ◽  
R Goonewardene ◽  
K N Mendis
Keyword(s):  
T Cells ◽  
Plasmodium Vivax ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Clinical Symptoms ◽  
Gastrointestinal Symptoms ◽  
Gamma Delta T Cells ◽  
Gamma Delta ◽  
Endemic Region ◽  
Gastrointestinal Pathology

The percentage of peripheral blood mononuclear cells (PBMC) bearing the CD3+ phenotype and the alpha/beta and gamma/delta T cell receptors (TCR) in PBMC were examined in Plasmodium vivax malaria patients and convalescents. The cells were labeled with monoclonal antibodies, stained with either fluorescence or phycoerythrin, and examined by ultraviolet (UV) microscopy. A highly significant increase in both the proportion and the absolute numbers of gamma/delta T cells (p &lt; 0.005 and &lt; 0.001, respectively, Student's t test) was observed in nonimmune P. vivax patients during clinical paroxysms compared to nonmalarial controls. These T cells, which normally constitute not more than 3-5% of PBMC, constituted &lt; or = to 30% of PBMC during paroxysms in these nonimmune patients in whom the clinical symptoms were severe. A less significant increase of gamma/delta T cells were also observed in these nonimmune patients during infection, between paroxysms and during convalescence. In contrast, in an age-matched group of semi-immune patients resident in a malaria-endemic region of the country, in whom the clinical disease was comparatively mild, there was no increase in gamma/delta T cells either during infection, even during paroxysms, or convalescence. The severity of disease symptoms in patients as measured by a clinical score correlated positively with the proportion of gamma/delta T cells in peripheral blood (r = 0.53, p &lt; 0.01), the most significant correlation being found between the prevalence and severity of gastrointestinal symptoms, nausea, anorexia, and vomiting, and the proportion of gamma/delta T cells (r = 0.49, p = 0.002). These findings suggest that gamma/delta T cells have a role to play in the pathogenesis of malaria, possibly in the general constitutional disturbances and particularly in gastrointestinal pathology in malaria.

scholarly journals Analysis of interferon-inducible genes in cells of chronic myeloid leukemia patients responsive or resistant to an interferon-alpha treatment

Blood ◽  
1990 ◽  
Vol 76 (11) ◽  
pp. 2337-2342
Author(s):  
IM Clauss ◽  
B Vandenplas ◽  
MG Wathelet ◽  
C Dorval ◽  
A Delforge ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Chronic Myeloid Leukemia ◽  
Interferon Alpha ◽  
Peripheral Blood ◽  
Myeloid Leukemia ◽  
Bone Marrow Cells ◽  
Healthy Controls ◽  
Ifn Alpha ◽  
Inducible Genes ◽  
Marrow Cells

Recombinant human interferon-alpha (IFN-alpha) can induce a hematologic remission in patients with chronic myeloid leukemia. However, some patients are resistant and others develop late resistance to the IFN- alpha treatment. To understand the molecular mechanism of this resistance, we have analyzed the expression of 10 IFN-inducible genes in the cells of three resistant patients, two responsive patients, and six healthy controls. Northern blot hybridizations showed that all the genes were induced in in vitro IFN-alpha treated peripheral blood cells of the patients and healthy controls. These genes were also inducible in peripheral blood and bone marrow cells of two out of two resistant patients administered an injection of IFN-alpha. We conclude that the resistance to the IFN-alpha treatment of the chronic myeloid leukemia patients we studied is not due to (1) the absence of induction of any of the 10 IFN-inducible genes we studied, including the low-molecular- weight 2′-5′oligoadenylate synthetase; (2) the presence of an antagonist of IFN-alpha in the peripheral blood or bone marrow cells; and (3) the presence of neutralizing anti-IFN-alpha antibodies.

scholarly journals Isolation and characterization of (gamma, delta) CD4+ T cell clones derived from human fetal liver cells.

1989 ◽  
Vol 170 (3) ◽  
pp. 1009-1014 ◽  
Author(s):  
P Aparicio ◽  
J M Alonso ◽  
M L Toribio ◽  
M A Marcos ◽  
L Pezzi ◽  
...  
Keyword(s):  
T Cells ◽  
Fetal Liver ◽  
Physiological Role ◽  
Gamma Delta T Cells ◽  
Gamma Delta ◽  
Human Fetal Liver ◽  
Isolation And Characterization ◽  
New Information ◽  
Cell Clones

Lymphocytes isolated from human fetal liver and expanded in vitro in IL-2-containing media reveal the existence of CD4+ gamma, delta T cells. These cells display differential features of double-negative and CD8+ gamma, delta T cells as well as of CD4+ alpha, beta T cells. Thus, they failed to lyse targets in lectin-mediated killing assays and to perform classical helper functions. These results add new information necessary for a better understanding of the physiological role of the gamma, delta T cells.

Allogeneic Vδ1 gamma delta T cells engineered with glypican-3 (GPC3)-specific CAR expressing soluble IL-15 have enhanced antitumor efficacy against hepatocellular carcinoma in preclinical models.

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. e14511-e14511
Author(s):  
Amani Makkouk ◽  
Xue (Cher) Yang ◽  
Taylor Barca ◽  
Anthony Lucas ◽  
Mustafa Turkoz ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
T Cells ◽  
T Cell ◽  
Healthy Donor ◽  
Cytokine Production ◽  
Tumor Control ◽  
Gamma Delta ◽  
T Cell Therapy ◽  
Donor Pbmcs

e14511 Background: Autologous αβ chimeric antigen receptor (CAR) T cell therapy has shown promising clinical results in hematologic malignancies but limited success in solid tumors. Allogeneic αβ T cell therapy may overcome several challenges faced by autologous therapy but carries the risk of graft-versus-host disease (GvHD) and does not readily recognize multiple tumor-associated antigens. Gamma delta (γδ) T cells are highly cytolytic effectors that can recognize and kill tumor cells in an MHC-unrestricted manner without causing GvHD. The Vδ1 subset is preferentially localized in peripheral tissue and is critical for tumor immunosurveillance. Engineering Vδ1 T cells with CARs can further enhance antitumor activity and represents an attractive and safe approach to treating solid tumors. However, their clinical use has been hindered by the limited number of circulating Vδ1 T cells. Here, we describe the development of the first allogeneic Vδ1 T cells that have been expanded from healthy donor PBMCs and genetically modified to secrete IL-15 (sIL15) and express a CAR targeting glypican-3 (GPC3), a rational target for hepatocellular carcinoma (HCC). Methods: Vδ1 T cells in healthy donor PBMCs were activated by a Vδ1-specific monoclonal antibody and transduced with 41BBζ or 41BBζ-sIL15 GPC3-CARs prior to cell expansion, αβ T cell depletion and cryopreservation. In vitro characterization included: 1) co-culture assays with GPC3-expressing HCC targets HepG2 and PLC/PRF/5, 2) phenotypic analysis by flow cytometry, and 3) cytokine production by multiplexed immunoassay. For in vivo assessment of tumor control, immunodeficient NSG mice were subcutaneously injected with HepG2 cells and treated with a single dose of 41BBζ or 41BBζ-sIL15 GPC3-CAR Vδ1 T cells. Additionally, tissues were harvested 7 days post transfer and analyzed by flow cytometry for Vδ1 T cell tissue homing and proliferation, or at end of study and analyzed for GvHD by immunohistochemistry. Results: Vδ1 T cells expanded over 10,000-fold and routinely reached >80% purity. Expanded Vδ1 T cells showed a primarily naïve-like phenotype (CD45RA+CD27+) with minimal exhaustion receptor expression and displayed robust proliferation, cytokine production, and cytotoxic activity against HCC cell lines expressing low and high GPC3 levels in vitro. In a HepG2 mouse model, GPC3-CAR Vδ1 T cells primarily accumulated and proliferated in the tumor, and a single dose was able to efficiently control tumor burden without causing GvHD. Importantly, 41BBζ-sIL15 GPC3-CAR Vδ1 cells displayed enhanced tumor-specific proliferation that resulted in better tumor control without any toxicity. Conclusions: Our results show that expanded Vδ1 T cells engineered with GPC3-CAR and sIL-15 represent a promising platform for safe and effective off-the-shelf treatment of HCC.

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