scholarly journals iPSC-Derived NK Cells and Anti-PD-1 Antibody Synergize to Enhance T Cell Cytokine and Cytolytic Responses Against Multiple Tumors

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 730-730
Author(s):  
Frank Cichocki ◽  
Ryan Bjordahl ◽  
Svetlana Gaidarova ◽  
Sajid Mahmood ◽  
Paul Rogers ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Nk Cells ◽  
Nk Cell ◽  
K562 Cells ◽  
Tumor Spheroid ◽  
Activated T Cells ◽  
Cell Responses ◽  
T Cell Migration ◽  
Tumor Reduction

Abstract The development of immunotherapeutic monoclonal antibodies targeting checkpoint inhibitory receptors (CIR), such as programmed death 1 (PD-1), has transformed the oncology landscape. However, many tumor subtypes are resistant to CIR-targeted therapy, and relapse remains a significant concern. Therefore, combination of novel immunotherapies with CIR targeting remains a promising and widely investigated approach to bolster anti-tumor responses and to overcome tumor resistance to CIR therapy. Natural killer (NK) cells mediate direct tumor cell lysis and are key regulators of T cell responses through the production of inflammatory cytokines and chemokines. In many cancers, NK cell numbers are low and their functional responses are sub-optimal. The use of allogeneic NK cell immunotherapy has shown significant clinical promise for the treatment of acute myelogenous leukemia (AML). However, this approach has inherent limitations with respect to the number of NK cells that can be isolated and variability in the quantity and quality of NK cells between donors. To overcome these barriers, we have developed a system for large scale expansion of NK cells derived from induced pluripotent stem cells (iPSCs) to be combined with CIR antibodies for multiple tumor types (Figure 1A). iPSC derive NK (iNK) cells (defined as CD45+CD3-CD56+) differentiated with high efficiency in this culture system (Figure 1B), and overall expansion from the hematopoietic progenitor stage to end of the protocol was approximately 1 million-fold (Figure 1C). iNK cells displayed a cell surface phenotype typical of primary peripheral blood NK cells, with high expression of the cytotoxicity molecules granzyme B and perforin. Functionally, iNK cells degranulated and produced the pro-inflammatory cytokines tumor necrosis factor (TNF) and interferon (IFN)-γ at high frequencies in response to K562 cells (a myeloid leukemia cell line) (Figure 1D). These cells also effectively killed a broad range of solid tumor targets, including ovarian cancer (SKOV-3), lung cancer (A549) and pancreatic cancer (PANC-1) cell lines, in 2D tissue culture assays (Figure 1E). To interrogate the ability of iPSC-derived NK cells to synergize with CIR therapy, we used an in vitro 3D tumor spheroid system to model the combinatorial effects of T cells, iNK cells, and checkpoint blockade in the context of anti-tumor function. Using SKOV-3 spheroids as targets in a 160-hour killing assay, we found that iNK cells could mediate significant, but not complete destruction of tumor spheroids (46% tumor reduction). Addition of twice as many activated T cells by themselves induced tumor spheroid destruction (58% tumor reduction). The combination of iNK and activated T cells led to robust target cell destruction (71% tumor reduction). Importantly, the combination of activated T cells, iNK cells and anti-PD-1 antibody led to a near complete elimination of tumor spheroid targets, with greater than 99% tumor reduction (Figure 1F). In addition to synergistic lysis of tumor spheroids, analyses of cytokine secretion in iNK, activated T cell and CIR spheroid cultures revealed synergistic production of TNF and IFN-g (Figure 1G). The results from our 3D spheroid assays led us to hypothesize that iNK cells could recruit T cells to promote tumor lysis. To test this hypothesis, we performed transwell migration assays to test the relative abilities of iNK cells and K562 cells to promote T cell migration. We found that, relative to media controls, both iNK cells and K562 cells promoted T cell migration across transwells, and the highest amount of degree of migration was observed in the presence of both iNK and K562 cells (Figure 1H). Finally, we tested iNK-mediated T cell recruitment in an in vivo setting where iNK cells and IL-2 were injected with or without K562 cells into the peritoneal cavities of immune-deficient NSG mice, and activated T cells were injected retro-orbitally into the blood. Mice were sacrificed 4 days later, and total T cells in the peritoneal cavities were determined. Compared to the T cell only control group, iNK cells promoted significant T cell recruitment to the peritoneum, which was amplified upon iNK cell activation with the addition of K562 cells (Figure 1I). Together, our data demonstrate that iNK cells can serve as an off-the-shelf source of NK cells and have the potential to synergize with anti-PD-1 CIR therapy to enhance anti-tumor T cell responses. Disclosures Cichocki: Fate Therapeutics Inc.: Consultancy, Research Funding. Bjordahl:Fate Therapeutics Inc.: Employment. Gaidarova:Fate Therapeutics Inc: Employment. Mahmood:Fate Therapeutics Inc.: Employment. Rogers:Fate Therapeutics Inc: Employment. Ge:Fate Therapeutics Inc.: Employment. Kaufman:Fate Therapeutics: Consultancy, Research Funding. Valamehr:Fate Therapeutics Inc.: Employment.

scholarly journals The Role of DNAM-1/Tigit Axis of NK Cells in the Regulation of Alloreactive T Cell Responses and the Potential Mechanism

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4530-4530
Author(s):  
Lixia Sheng ◽  
Guifang Ouyang ◽  
MU Qitian ◽  
He Huang
Keyword(s):  
T Cells ◽  
T Cell ◽  
Nk Cells ◽  
Nk Cell ◽  
Activated T Cells ◽  
Over Expression ◽  
Lentiviral Transduction ◽  
Cell Responses ◽  
Alloreactive T Cell

Abstract Objective: Previous studies has showed the important role of NK cell in the regulation of alloreactive T cell response and negative regulation of GVHD. The paired receptors DNAM-1 and TIGIT, which bind the same ligands but have opposite effects on NK cell function, might present as potential targets for the GVHD treatment. This study is designed to explore the role of TIGIT/DNAM-1 balance of NK cells in the regulation of alloreactive T cell responses and the potential mechanism. Methods: Antibodies blocking of TIGIT or DNAM-1, over-expression of TIGIT or DNAM-1 via lentiviral transduction and knockdown of TIGIT or DNAM-1 by lentiviral shRNA were used to manipulate the TIGIT/DNAM-1 balance on NK cell. Cytotoxicity assay using alloantigen activated T cells as targets were used to evaluate the regulating function of NK cell on alloreactive T cell responses. Western blot and small molecule inhibitors against PI3K were combined to investigate whether the PI3K-Akt-ERK signaling cascade is involved in the signal transduction process following TIGIT/DNAM-1-PVR engagement. RESULTS: Blocking of DNAM-1 by an anti-DNAM-1 antibody and knockdown of DNAM-1 expression by lentiviral shRNA both resulted in deceased cytotoxicity of NK cells against alloantigen activated T cells, while over-expression of DNAM-1 via lentiviral transduction resulted in enhanced cytotoxicity. Blocking of TIGIT by an anti-TIGIT antibody and knockdown of TIGIT expression by lentiviral shRNA both resulted in increased cytotoxicity of NK cells against alloantigen activated T cells, while over-expression of TIGIT via lentiviral transduction resulted in decreased cytotoxicity. Increases in NK cytotoxicity against activated T cells through TIGIT knockdown could be overcome by blocking DNAM-1 signaling. Simultaneously, over-expression of DNAM-1 or knockdown of TIGIT expression resulted in an increase of the phosphorylation levels of Akt and ERK1/2 in NK cells after contacted with activated T cells, which could be overcome by pretreating NK cells with anti-DNAM-1 or PI3K small molecule inhibitor. Pretreating alloantigen activated T cells with anti-PVR also resulted in deceased cytotoxicity and Akt and ERK1/2 phosphorylation in DNAM-1 over-expression NK cells. Conclusion: The paired receptor DNAM-1/TIGIT on the surface of NK cells compete the same PVR ligand on the surface of activated T cells and the DNAM-1/TIGIT axis is involved in the regulation of cytotoxicity of NK cells on alloantigen activated T cells through PI3K-Akt-ERK cascade phosphorylation. The DNAM-1/TIGIT expression balance may present as biomarkers for aGVHD and potential targets for aGVHD therapy. Disclosures No relevant conflicts of interest to declare.

Antigen-activated human T lymphocytes express cell-surface NKG2D ligands via an ATM/ATR-dependent mechanism and become susceptible to autologous NK- cell lysis

Blood ◽  
2007 ◽  
Vol 110 (2) ◽  
pp. 606-615 ◽  
Author(s):  
Cristina Cerboni ◽  
Alessandra Zingoni ◽  
Marco Cippitelli ◽  
Mario Piccoli ◽  
Luigi Frati ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Lymphocytes ◽  
T Cell Activation ◽  
Cell Activation ◽  
Nk Cell ◽  
Protein A ◽  
T Cell Responses ◽  
Activated T Cells ◽  
Cell Responses

AbstractRecent evidence indicates that natural killer (NK) cells can negatively regulate T-cell responses, but the mechanisms behind this phenomenon as a consequence of NK–T-cell interactions are poorly understood. We studied the interaction between the NKG2D receptor and its ligands (NKG2DLs), and asked whether T cells expressed NKG2DLs in response to superantigen, alloantigen, or a specific antigenic peptide, and if this rendered them susceptible to NK lysis. As evaluated by FACS, the major histocompatibility complex (MHC) class I chain-related protein A (MICA) was the ligand expressed earlier on both CD4+ and CD8+ T cells in 90% of the donors tested, while UL16-binding protein-1 (ULBP)1, ULBP2, and ULBP3 were induced at later times in 55%–75% of the donors. By carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling, we observed that NKG2DLs were expressed mainly on T cells that had gone through at least one division. Real-time reverse-transcription polymerase chain reaction confirmed the expression of all NKG2DLs, except ULBP4. In addition, T-cell activation stimulated phosphorylation of ataxia-telangiectasia mutated (ATM), a kinase required for NKG2DLs expression after DNA damage, and ATM/Rad3-related kinase (ATR) inhibitors blocked MICA induction on T cells with a mechanism involving NF-κB. Finally, we demonstrated that activated T cells became susceptible to autologous NK lysis via NKG2D/NKG2DLs interaction and granule exocytosis, suggesting that NK lysis of T lymphocytes via NKG2D may be an additional mechanism to limit T-cell responses.

Natural Killer Cell Licensing Delineates NK “Helper/Repair” and NK “Effector/Suppressor” Subsets During Viral Infections

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 13-13
Author(s):  
Can M. Sungur ◽  
Anthony E. Zamora ◽  
Ethan G. Aguilar ◽  
Yajarayma Tang-Feldman ◽  
Juan Du ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Lymph Nodes ◽  
Viral Infection ◽  
Nk Cells ◽  
Nk Cell ◽  
Cell Subset ◽  
Gm Csf ◽  
Cell Responses ◽  
Late Stages

Abstract Natural killer (NK) cells are innate lymphocytes with anti-viral and anti-tumor capabilities that can be divided into subsets based on differential receptor expression patterns. NK cells that express inhibitory receptors that can bind to the MHC class I molecules present in the host are considered to be “licensed,” fully functional NK cells with normal production of cytokines and cytotoxicity in response to targets. In contrast, “unlicensed” NK cells are unable to strongly bind to host MHC class I molecules and are in turn hyporesponsive to targets in terms of cytotoxicity and cytokine production. Recent data suggest that NK cells also regulate antigen-specific adaptive immune responses during the course of viral infection, playing a significant role in viral clearance and immunopathology. The specific populations of NK cells that may mediate these differential effects during the course of viral infection have not been identified. Here, we demonstrate differential effector and immunoregulatory functions of licensed versus unlicensed NK cells during influenza and murine cytomegalovirus (MCMV) infections in mouse models. We hypothesize that licensed NK cells serve a dual role as both effector and suppressor populations depending on the stage of viral infection. Similarly, unlicensed NK cells serve a dual role as helper and repair populations during the early and late stages of viral infection, respectively. We performed licensed and unlicensed NK cell subset depletions and then infected mice with influenza or MCMV and ascertained effects on: viral titers, antigen-specific T cell responses, and tissue pathology. Our data show that after influenza or MCMV infection, there is a significant reduction in antigen-specific CD4+ and CD8+ T cell populations in the presence of licensed NK cells as determined by tetramer-positive cells. Targeting of these T cells by the NK “effector/suppressor” licensed population appears later in the time course of infection and to be through NKG2D recognition and perforin-mediated lysis based on upregulation of NKG2D ligands Rae-1 and MULT1 on the T cells and the loss of T cell regulation with NKG2D blockade or perforin knockout mice. Depletion of the unlicensed NK “helper/repair” subset reduced the number of DCs in the lymph nodes and reduced total antigen-specific T cells. The unlicensed NK cells were found to home to the lymph node and produce increased levels of GM-CSF early during infection resulting in DC expansion. Additionally, the unlicensed NK cells are the primary producers of IL-22 based on intracellular staining in the damaged tissues in the late stages of viral infection, aiding in tissue regeneration. Adoptive transfer of unlicensed NK cells with IL-22 silencing through siRNA transfection into immunodeficient mice showed increased tissue damage and pathology as compared to transfer of non-IL-22 silenced NK cells. Collectively, these data suggest differential roles of licensed versus unlicensed NK cells that are both tissue and time-specific. At early stages of infection, licensed NK cells serve as direct anti-viral cells at the sites of infection while unlicensed cells promote DC expansion in the lymph nodes promoting antigen-specific T cell responses. Conversely, at the late stages of infection, licensed NK cells serve an immunoregulatory role by lysing antigen-specific T cells at the site of infection and in the lymph nodes, while unlicensed NK cells travel to the sites of injury to aid in tissue repair through production of IL-22. Importantly, a similar functional polarization of resting human NK cells was also observed after PMA/ionomycin stimulation, with a small population of unlicensed NK cells producing IL-22 and a bias towards GM-CSF secretion over IFNγ production by the unlicensed NK cell subset. We conclude that licensed NK cells have an effector/suppressor function while the unlicensed NK cells function as the helper/repair population suggesting distinct roles of NK cell subsets throughout the course of infection. By understanding the functions and characteristics of these NK cell populations, specific subsets can either by adoptively transferred or therapeutically targeted clinically to aid in different stages of immunological response including elimination of the virus, inhibiting the adaptive immune response, or aiding in tissue repair and regeneration. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Adenovirus Vector Vaccination Impacts NK Cell Rheostat Function following Lymphocytic Choriomeningitis Virus Infection

2018 ◽  
Vol 92 (11) ◽  
Author(s):  
Eryn Blass ◽  
Malika Aid ◽  
Amanda J. Martinot ◽  
Rafael A. Larocca ◽  
Zi Han Kang ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Nk Cells ◽  
Nk Cell ◽  
Adenovirus Vector ◽  
T Cell Responses ◽  
Lymphocytic Choriomeningitis ◽  
Cell Depletion ◽  
Cell Responses ◽  
Lcmv Infection

ABSTRACT Natural killer (NK) cells respond rapidly as a first line of defense against infectious pathogens. In addition, NK cells may provide a “rheostat” function and have been shown to reduce the magnitude of antigen-specific T cell responses following infection to avoid immunopathology. However, it remains unknown whether NK cells similarly modulate vaccine-elicited T cell responses following virus challenge. We used the lymphocytic choriomeningitis virus (LCMV) clone 13 infection model to address whether NK cells regulate T cell responses in adenovirus vector-vaccinated mice following challenge. As expected, NK cell depletion in unvaccinated mice resulted in increased virus-specific CD4 + and CD8 + T cell responses and immunopathology following LCMV challenge. In contrast, NK cell depletion had minimal to no impact on antigen-specific T cell responses in mice that were vaccinated with an adenovirus serotype 5 (Ad5)-GP vector prior to LCMV challenge. Moreover, NK cell depletion in vaccinated mice prior to challenge did not result in immunopathology and did not compromise protective efficacy. These data suggest that adenovirus vaccine-elicited T cells may be less sensitive to NK cell rheostat regulation than T cells primed by LCMV infection. IMPORTANCE Recent data have shown that NK cell depletion leads to enhanced virus-elicited T cell responses that can result in severe immunopathology following LCMV infection in mice. In this study, we observed that NK cells exerted minimal to no impact on vaccine-elicited T cells following LCMV challenge, suggesting that adenovirus vaccine-elicited T cells may be less subject to NK cell regulation. These data contribute to our understanding of NK cell regulatory functions and T cell-based vaccines.

scholarly journals iPSC-Derived NK Cells Synergize with T Cells and Anti-PD-1 Antibody to Mediate Durable Anti-Tumor Responses In Vivo

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1933-1933
Author(s):  
Jeffrey S. Miller ◽  
Ryan Bjordahl ◽  
Svetlana Gaidarova ◽  
Sajid Mahmood ◽  
Paul Rogers ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Nk Cells ◽  
Board Of Directors ◽  
Research Funding ◽  
Tumor Control ◽  
Tumor Spheroid ◽  
Advisory Committees ◽  
Tumor Reduction

Cancer immunotherapies have resulted in a paradigm shift in therapy. One of the most successful approaches has been administration of antibodies targeting immune checkpoint inhibitors (ICI), such as programmed death-1 (PD-1), that can reinvigorate functionally exhausted T cells. Unfortunately, durable tumor regression is limited to a minority of patients, and relapse remains a significant concern. Combining novel immunotherapies with ICI s a promising strategy to bolster antitumor responses and response rates. Natural killer (NK) cells mediate direct tumor cell lysis, effectively target MHC low or null transformed cells and are key regulators of T cell responses through the production of inflammatory cytokines and chemokines. In many cancers, NK cell numbers are low, and their functional responses are sub-optimal. The use of allogeneic peripheral blood NK cells for immunotherapy has shown significant clinical promise for the treatment of various cancers. However, sourcing NK cells for adoptive cell therapy has been limited by both cell number and quality. Thus, we developed a robust manufacturing system for the differentiation and expansion of high-quality NK cells derived from induced pluripotent stem cells (iPSCs) that can be combined with ICI antibodies for multiple tumor types. To interrogate the ability of iPSC-derived NK cells to synergize with ICI therapy, we developed an in vitro 3D tumor spheroid system to model the combinatorial effects of activated CD3+ T cells, iPSC-derived NK (iNK) cells and ICI blockade for anti-tumor function in a more physiological context than the standard 2D cultures and in real time. Using SKOV-3 (an ovarian cancer line) spheroids as targets in a 160-hour killing assay, we found that iNK cells could mediate significant, but not complete destruction of tumor spheroids (46% tumor reduction). Twice as many activated CD3+ T cells by themselves also induced significant but incomplete tumor spheroid destruction (58% tumor reduction). Combined iNK and activated CD3+ T cells led to robust target cell destruction (71% tumor reduction). Importantly, adding anti-PD-1 antibody (mAb) to activated CD3+ T cells and iNK cells led to near complete elimination of tumor spheroid targets, with >99% tumor reduction. Cytokine and chemokine secretion analyses in co-cultures of activated CD3+ T cells and iNK cells revealed synergistic production of CCL3 and CCL4 for T cell recruitment, and TNF and IFN-γ to augment anti-tumor responses. To determine whether these striking 3D tumor spheroid results simulate the in vivo setting, we injected luciferase-expressing OVCAR8 (a human ovarian cancer line) cells into the peritoneal cavities of immunodeficient NSG mice. Following sublethal irradiation, we treated groups of mice with either anti-PD-1 mAb, activated CD3+ T cells or iNK cells alone or in various combinations. IL-2 was injected i.p. twice weekly for two weeks into all mice except those in the tumor alone group (Figure 1A). In mice that received iNK cells alone, significant tumor control was observed over the first 21 days but was not sustained. Tumor control was similar between groups of mice that received iNK cells and mice that received iNK cells and anti-PD-1 mAb. Mice treated with either anti-PD-1 mAb or activated CD3+ T cells alone exhibited similar rates of tumor growth relative to the untreated group. Modest tumor control was observed in the group of mice that received combined activated CD3+ T cells and anti-PD-1 mAb, though the effect did not reach statistical significance. Durable tumor control past day 21 was observed in groups of mice that received either combined activated CD3+ T cells and iNK cells or activated CD3+ T cells + iNK cells with anti-PD-1 mAb (Figure 1B). Importantly, 50% of mice in the activated CD3+ T cells + iNK cells + anti-PD-1 mAb group exhibited tumor bioluminescence readings well below other treatment group at day 35 (Figure 1C, D). Collectively, these data demonstrate that iNK cells can serve as an off-the-shelf source of high-quality NK cells and synergize with anti-PD-1 ICI therapy to enhance anti-tumor T cell responses in vitro and in vivo, providing a novel immunotherapeutic platform for tumors in which iNK cells, activated CD3+ T cells or anti-PD-1 mAb therapy alone is not sufficiently effective. The current program is under clinical investigation and can be found at clinicaltrials.gov NCT03841110. Disclosures Miller: Moderna: Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics, Inc: Consultancy, Research Funding; CytoSen: Membership on an entity's Board of Directors or advisory committees; OnKImmune: Membership on an entity's Board of Directors or advisory committees; Dr. Reddys Laboratory: Membership on an entity's Board of Directors or advisory committees; GT BioPharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Bjordahl:Fate Therapeutics, Inc.: Employment. Gaidarova:Fate Therapeutics, Inc: Employment. Mahmood:Fate Therapeutics, Inc: Employment. Rogers:Fate Therapeutics, Inc: Employment. Moyar:Fate Therapeutics, Inc: Employment. Blazar:Abbvie Inc: Research Funding; KidsFirst Fund: Research Funding; Childrens' Cancer Research Fund: Research Funding; Leukemia and Lymphoma Society: Research Funding; Regeneron Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Magenta Therapeutics and BlueRock Therapeuetics: Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics, Inc.: Research Funding; Tmunity: Other: Co-Founder; BlueRock Therapeutics: Membership on an entity's Board of Directors or advisory committees; RXi Pharmaceuticals: Research Funding; Alpine Immune Sciences, Inc.: Research Funding; Kamon Pharmaceuticals, Inc: Membership on an entity's Board of Directors or advisory committees; Five Prime Therapeutics Inc: Co-Founder, Membership on an entity's Board of Directors or advisory committees. Kaufman:FATE Therapeutics: Consultancy, Research Funding. Valamehr:Fate Therapeutics, Inc: Employment. Cichocki:Fate Therapeutics, Inc: Research Funding. OffLabel Disclosure: NK cells

Impaired NK Cells Cytotoxicity Against Activated, Alloreactive Donor T Cells in aGVHD and Ability of Dasatnib to Restored the Gvhd-Regulation Function of NK Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3906-3906
Author(s):  
Lixia Sheng ◽  
Huarui Fu ◽  
Yongxian Hu ◽  
Shan Fu ◽  
Yamin Tan ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Nk Cells ◽  
Nk Cell ◽  
Activated T Cells ◽  
Hematopoietic Stem ◽  
Alloreactive T Cells ◽  
Activating Receptors ◽  
Regulation Function ◽  
Nk Cytotoxicity

Abstract In murine models, donor natural killer cells(NK) exhibit immunoregulatory functions to alloreactive T cells during the initiation of acute graft versus host disease(aGVHD). The immunoregulatory role of NK cells in human aGVHD remains unclear. Here we compared the regulation of alloreactive donor T cell response by donor CD56+NK cells in 63 patients receiving allogeneic hematopoietic stem cell transplantation(allo-HSCT) and their donors. We found that NK cells from donors effectively suppressed T cell proliferation in response to Allo-DCs, showing cytotoxicity against activated proliferating T cells but not resting T cells. Subgroup of NK cells influenced the cytotoxicity against allo-reactive T cells, NKG2A-CD57+ NK cells degranulated to activated auto-T cells more potently than NKG2A+CD57- subgroup, suggesting NKG2A and CD57 expression patterns influenced NK cytotoxicity against activated T cells. When we analyzed the alteration in potential ligands for NK activating receptors on CD3+T cells during stimulated by allo-antigens, we found that activated T cells expressed higher levels of NKG2D-L(MICA/B,ULBP-1/ 2/ 4), DNAM1-L(PVR), and LFA-L(ICAM-1 and ICAM-2). Using neutralizing antibodies to block the interaction between NK receptors and correspondence ligands, we found that both activating receptor(LFA-1,NKG2D and DNAM-1) and inhibited receptor(NKG2A and TIM-3) participated this process. In the first 3 months post HSCT, reconstituted NK cells were mainly CD56bright and NKG2A+ CD57- subgroup, and percent of CD11b+CD27+ subgroup was significantly higher than in health donors, indicating relative immature subgroup predominated the early reconstituted NK cells after transplantation. By evaluating the dynamic restitution regularity of NK cell receptoires after Allo-HSCT, we found that the early reconstituted NK cells had a notably decreased surface expression of DNAM-1 and NKG2D compared with their corresponding donors. Furthermore, we compared the expression of receptors on CD56+NK cells from patients who developed aGVHD (group GVHD) with those without aGVHD (group non-GVHD) at 4 weeks after transplantation. Interestingly, we found that decreased expression of DNAM-1 and NKG2D and enhanced NKG2A expression are associated with aGVHD. When we assessed the expression of ligands for activating NK-cell receptors on activated T cells in aGVHD and non-aGVHD patients, we found that T cells in aGVHD patients expressed higher level of PVR(ligand for DNAM-1) and MICA/B(ligand for NKG2D) when compared with no-aGVHD patients or donors. To explore whether the subgroup alteration and reduced activating receptors expression on NK cells in aGVHD patients affected their capacity of GVHD regulation, we next examined NK-cell degranulation and cytotoxicity to allogeneic antigen activated T cells. The results demonstrated that the ability of donor NK cells to inhibit and lyse autologous activated T cells is impaired during human GVHD. Of clinical relevance, the tyrosine kinase inhibitor(TKI) dasatinib enhanced NK cytotoxicity towards activated T cells by up-regulating the expression of CD226 and NKG2D and enhancing the proportion of CD57+NKG2A- subgroup. This study demonstrates for the first time that the ability of donor NK cells to inhibit alloreactive T cells response is impaired during human GVHD and dasatinib may reinforced the GVHD-regulation function of NK cells, which potentially may provide an opportunity for therapeutic treatment of GVHD. Disclosures No relevant conflicts of interest to declare.

scholarly journals Anti-tumor effects of RTX-240: an engineered red blood cell expressing 4-1BB ligand and interleukin-15

2021 ◽  
Author(s):  
Shannon L. McArdel ◽  
Anne-Sophie Dugast ◽  
Maegan E. Hoover ◽  
Arjun Bollampalli ◽  
Enping Hong ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Blood Cell ◽  
Nk Cells ◽  
Red Blood Cell ◽  
Safety Profile ◽  
Nk Cell ◽  
Cell Activity ◽  
In Vivo Studies

AbstractRecombinant agonists that activate co-stimulatory and cytokine receptors have shown limited clinical anticancer utility, potentially due to narrow therapeutic windows, the need for coordinated activation of co-stimulatory and cytokine pathways and the failure of agonistic antibodies to recapitulate signaling by endogenous ligands. RTX-240 is a genetically engineered red blood cell expressing 4-1BBL and IL-15/IL-15Rα fusion (IL-15TP). RTX-240 is designed to potently and simultaneously stimulate the 4-1BB and IL-15 pathways, thereby activating and expanding T cells and NK cells, while potentially offering an improved safety profile through restricted biodistribution. We assessed the ability of RTX-240 to expand and activate T cells and NK cells and evaluated the in vivo efficacy, pharmacodynamics and tolerability using murine models. Treatment of PBMCs with RTX-240 induced T cell and NK cell activation and proliferation. In vivo studies using mRBC-240, a mouse surrogate for RTX-240, revealed biodistribution predominantly to the red pulp of the spleen, leading to CD8 + T cell and NK cell expansion. mRBC-240 was efficacious in a B16-F10 melanoma model and led to increased NK cell infiltration into the lungs. mRBC-240 significantly inhibited CT26 tumor growth, in association with an increase in tumor-infiltrating proliferating and cytotoxic CD8 + T cells. mRBC-240 was tolerated and showed no evidence of hepatic injury at the highest feasible dose, compared with a 4-1BB agonistic antibody. RTX-240 promotes T cell and NK cell activity in preclinical models and shows efficacy and an improved safety profile. Based on these data, RTX-240 is now being evaluated in a clinical trial.

scholarly journals Novel self-amplificatory loop between T cells and tenocytes as a driver of chronicity in tendon disease

2021 ◽  
pp. annrheumdis-2020-219335
Author(s):  
Emma Garcia-Melchor ◽  
Giacomo Cafaro ◽  
Lucy MacDonald ◽  
Lindsay A N Crowe ◽  
Shatakshi Sood ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Migration ◽  
T Cell ◽  
Inflammatory Cytokines ◽  
T Cell Activation ◽  
Cell Activation ◽  
Conditioned Media ◽  
Tissue Remodelling ◽  
Activated T Cells ◽  
T Cell Migration

ObjectivesIncreasing evidence suggests that inflammatory mechanisms play a key role in chronic tendon disease. After observing T cell signatures in human tendinopathy, we explored the interaction between T cells and tendon stromal cells or tenocytes to define their functional contribution to tissue remodelling and inflammation amplification and hence disease perpetuation.MethodsT cells were quantified and characterised in healthy and tendinopathic tissues by flow cytometry (FACS), imaging mass cytometry (IMC) and single cell RNA-seq. Tenocyte activation induced by conditioned media from primary damaged tendon or interleukin-1β was evaluated by qPCR. The role of tenocytes in regulating T cell migration was interrogated in a standard transwell membrane system. T cell activation (cell surface markers by FACS and cytokine release by ELISA) and changes in gene expression in tenocytes (qPCR) were assessed in cocultures of T cells and explanted tenocytes.ResultsSignificant quantitative differences were observed in healthy compared with tendinopathic tissues. IMC showed T cells in close proximity to tenocytes, suggesting tenocyte–T cell interactions. On activation, tenocytes upregulated inflammatory cytokines, chemokines and adhesion molecules implicated in T cell recruitment and activation. Conditioned media from activated tenocytes induced T cell migration and coculture of tenocytes with T cells resulted in reciprocal activation of T cells. In turn, these activated T cells upregulated production of inflammatory mediators in tenocytes, while increasing the pathogenic collagen 3/collagen 1 ratio.ConclusionsInteraction between T cells and tenocytes induces the expression of inflammatory cytokines/chemokines in tenocytes, alters collagen composition favouring collagen 3 and self-amplifies T cell activation via an auto-regulatory feedback loop. Selectively targeting this adaptive/stromal interface may provide novel translational strategies in the management of human tendon disorders.

scholarly journals DNAM-1 promotes activation of cytotoxic lymphocytes by nonprofessional antigen-presenting cells and tumors

2008 ◽  
Vol 205 (13) ◽  
pp. 2965-2973 ◽  
Author(s):  
Susan Gilfillan ◽  
Christopher J. Chan ◽  
Marina Cella ◽  
Nicole M. Haynes ◽  
Aaron S. Rapaport ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Nk Cells ◽  
Adhesion Molecules ◽  
Cd8 T Cells ◽  
Nk Cell ◽  
Cd8 T Cell ◽  
Antigen Presenting

Natural killer (NK) cells and CD8 T cells require adhesion molecules for migration, activation, expansion, differentiation, and effector functions. DNAX accessory molecule 1 (DNAM-1), an adhesion molecule belonging to the immunoglobulin superfamily, promotes many of these functions in vitro. However, because NK cells and CD8 T cells express multiple adhesion molecules, it is unclear whether DNAM-1 has a unique function or is effectively redundant in vivo. To address this question, we generated mice lacking DNAM-1 and evaluated DNAM-1–deficient CD8 T cell and NK cell function in vitro and in vivo. Our results demonstrate that CD8 T cells require DNAM-1 for co-stimulation when recognizing antigen presented by nonprofessional antigen-presenting cells; in contrast, DNAM-1 is dispensable when dendritic cells present the antigen. Similarly, NK cells require DNAM-1 for the elimination of tumor cells that are comparatively resistant to NK cell–mediated cytotoxicity caused by the paucity of other NK cell–activating ligands. We conclude that DNAM-1 serves to extend the range of target cells that can activate CD8 T cell and NK cells and, hence, may be essential for immunosurveillance against tumors and/or viruses that evade recognition by other activating or accessory molecules.

scholarly journals Interferon Alpha Enhances NK Cell Function and the Suppressive Capacity of HIV-Specific CD8+T Cells

2018 ◽  
Vol 93 (3) ◽  
Author(s):  
Abena K. R. Kwaa ◽  
Chloe A. G. Talana ◽  
Joel N. Blankson
Keyword(s):  
T Cells ◽  
T Cell ◽  
Nk Cells ◽  
Effector Cell ◽  
Cell Function ◽  
Nk Cell ◽  
Suppressive Capacity ◽  
Short Course ◽  
Shock And Kill ◽  
Hiv 1

ABSTRACTCurrent shock-and-kill strategies for the eradication of the HIV-1 reservoir have resulted in blips of viremia but not in a decrease in the size of the latent reservoir in patients on suppressive antiretroviral therapy (ART). This discrepancy could potentially be explained by an inability of the immune system to kill HIV-1-infected cells following the reversal of latency. Furthermore, some studies have suggested that certain latency-reversing agents (LRAs) may inhibit CD8+T cell and natural killer (NK) cell responses. In this study, we tested the hypothesis that alpha interferon (IFN-α) could improve the function of NK cells from chronic progressors (CP) on ART. We show here that IFN-α treatment enhanced cytokine secretion, polyfunctionality, degranulation, and the cytotoxic potential of NK cells from healthy donors (HD) and CP. We also show that this cytokine enhanced the viral suppressive capacity of NK cells from HD and elite controllers or suppressors. Furthermore, IFN-α enhanced global CP CD8+T cell cytokine responses and the suppressive capacity of ES CD8+T cells. Our data suggest that IFN-α treatment may potentially be used as an immunomodulatory agent in HIV-1 cure strategies.IMPORTANCEData suggest that HIV+individuals unable to control infection fail to do so due to impaired cytokine production and/cytotoxic effector cell function. Consequently, the success of cure agendas such as the shock-and-kill strategy will probably depend on enhancing patient effector cell function. In this regard, NK cells are of particular interest since they complement the function of CD8+T cells. Here, we demonstrate the ability of short-course alpha interferon (IFN-α) treatments to effectively enhance such effector functions in chronic progressor NK cells without inhibiting their general CD8+T cell function. These results point to the possibility of exploring such short-course IFN-α treatments for the enhancement of effector cell function in HIV+patients in future cure strategies.

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