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.

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.

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.

Induction of a Novel Population of CD8+ Foxp3+ Regulatory T Cells During Graft Versus Host Disease

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 821-821
Author(s):  
Amy Beres ◽  
Dipica Haribhai ◽  
Chelsea Tessler-Verville ◽  
Patrick Gonyo ◽  
Martin Hessner ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Spleen Cells ◽  
Post Transplantation ◽  
Graft Versus Host ◽  
Cell Responses ◽  
Alloreactive T Cell

Abstract Abstract 821 Regulatory T cells defined as CD4+ and expressing the transcription factor Foxp3 have been shown to play a pivotal role in mitigating the severity of graft versus host disease (GVHD). In the course of studies designed to define the functional role of various CD4+ Treg populations in GVHD biology, we identified a novel population of CD8+ T cells that expressed Foxp3 and were induced early during this disease. While this population has been reported in patients with autoimmune disorders, the role of CD8+ Foxp3+ T cells in GVHD is unknown. To delineate the significance of this observation, we performed studies in which lethally irradiated Balb/c [H-2d] mice were transplanted with bone marrow and spleen cells from C57BL/6J [H-2b] mice that carried an EGFP reporter gene linked to Foxp3 (Foxp3EGFP). Tissues (spleen, lung, liver and colon) were harvested 5, 7, 10, 14 and 21 days post transplantation to define the temporal kinetics and absolute numbers of CD8+ Tregs during acute GVHD. We observed that CD8+ Foxp3+ T cells were detectable as early as five days post transplantation and persisted for up to three weeks in all GVHD target tissues. This cell population was present in similar percentages and absolute numbers to CD4+ Tregs in these tissue sites which is noteworthy given that the CD4+ Treg pool is comprised of two populations (natural Tregs and induced Tregs) whereas the CD8 pool is made up almost exclusively of Tregs that are induced, since only a very small percentage of CD8+ T cells from normal mice (<1.0%) constitutively express Foxp3. To determine whether the induction of CD8+ Tregs was a function of MHC disparity, we performed similar transplant studies using murine models with varying degrees of MHC incompatibility. Notably, the relative and absolute number of CD8+ Tregs were much lower in an MHC-matched, minor antigen mismatched model of GVHD [B6→Balb.B], and were absent in a model where only three amino acids distinguish donor and recipient [B6→bm1], indicating a correlation between CD8+ iTreg generation and MHC disparity between donor and host. To confirm that in vivo-induced CD8+ Tregs were suppressive, CD8+ Foxp3+ and CD4+ Foxp3+ T cells were sorted from the spleen and liver of B6→Balb/c GVHD mice six days post transplantation and examined in standard MLC suppression assays. These studies revealed that in vivo-derived CD8+ and CD4+ Tregs equally suppressed alloreactive T cell responses. Phenotypic analysis of in vivo-differentiated CD8 iTregs revealed that these cells expressed many of the same cell surface molecules as CD4+ Tregs (e.g. GITR, CD25, CD103, CTLA-4). To determine if CD8+ Foxp3+ T cells could be induced in vitro and used as adoptive therapy for GVHD prevention, purified CD8+ Foxp3EGFP– T cells were cultured with anti-CD3/CD28 antibodies, TGF-β and IL-2 for 3 days. Under these conditions, ∼30% of cells are induced to become Foxp3+. Addition of in vitro-differentiated CD8+ iTregs to a standard MLC resulted in potent suppression which was equivalent to that observed with in vitro-differentiated CD4+ Tregs. To determine whether these cells were suppressive in vivo, in vitro-differentiated CD8+ iTregs were adoptively transferred at a 1:1 Treg: effector cell ratio into lethally irradiated Balb/c mice that also received B6.PL BM and spleen cells to induce GVHD. In vitro-derived CD8+ iTregs failed to protect mice from GVHD in comparison to animals transplanted without CD8+ iTregs. This was attributable to reduced survival and the loss of Foxp3 expression in vivo. Furthermore, approximately 30–50% of these cells reverted to a proinflammatory phenotype characterized by IFN-γ secretion, similar to what has been described for in vitro-differentiated CD4+ iTregs (Beres et al, Clin Can Res, 2011). Finally, microarray studies were performed to compare the gene signatures of in vitro versus in vivo-induced CD8+ Tregs. Ontological analysis revealed that there was a 3–16 fold increase in the transcription of cytokine (e.g. IL-10) and cytotoxic (granzyme A, perforin, granzyme B) pathway genes in in vivo versus in vitro-induced CD8+ Tregs, suggesting that the former Treg population may employ similar mechanisms of suppression as has been reported for CD4+ Tregs. In summary, these studies have identified a novel population of CD8+ Foxp3+ cells that are induced early during GVHD, are able to suppress alloreactive T cell responses, and constitute another regulatory T cell population that is operative in GVHD biology. Disclosures: No relevant conflicts of interest to declare.

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 Natural killer cells recruited into lymph nodes inhibit alloreactive T-cell activation through perforin-mediated killing of donor allogeneic dendritic cells

Blood ◽  
2008 ◽  
Vol 112 (3) ◽  
pp. 661-671 ◽  
Author(s):  
Sophie Laffont ◽  
Cyril Seillet ◽  
John Ortaldo ◽  
Jérôme D. Coudert ◽  
Jean-Charles Guéry
Keyword(s):  
T Cell ◽  
Lymph Nodes ◽  
Nk Cells ◽  
Nk Cell ◽  
T Cell Responses ◽  
Cd8 T Cell ◽  
Cd4 T Cell ◽  
Cell Responses ◽  
Alloreactive T Cell ◽  
Draining Lymph Nodes

Abstract Natural killer (NK)–cell alloreactivity is exploited in bone marrow transplantation to improve clinical outcome. Likewise, in solid organ transplantation, it has been recently shown that recipient NK cells may limit alloreactive T-cell responses through their capacity to prevent the persistence of graft-derived allogeneic dendritic cells (DCs). In a model of CD4+ T cell–mediated allogeneic skin graft rejection, we show that the absence of host NK-cell alloreactivity was characterized by enhanced expansion of alloreactive effector T lymphocytes, including Th2 cells, and massive eosinophilic infiltrates in the rejected tissues. In CD8+ T cell–deficient C57BL/6 (H-2b) recipients injected with allogeneic BALB/c (H-2d) DCs, we demonstrated that NK cells expressing the H-2Dd-specific Ly49D activating receptor were implicated in the regulation of alloreactive CD4+ T-cell responses. Moreover, we showed that Ly49D+ CD127− NK cells were recruited within DC draining lymph nodes and rapidly eliminated allogeneic H-2d DCs through the perforin pathway. In normal mice, we further demonstrated that NK cells by quickly eliminating allogeneic DCs strongly inhibited alloreactive CD8+ T-cell responses. Thus, NK cells act as early regulators of alloreactive T-cell priming in allotransplantation through their capacity to kill allogeneic DCs in draining lymph nodes.

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.

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 Non-Hematopoietic Lymphoid Stromal Cells Prime Alloreactive CD4+ T Cells in Acute Graft-Versus-Host Disease

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4421-4421
Author(s):  
Muhammad Shaikh ◽  
Maria Ulbrich ◽  
Juan Gamboa Vargas ◽  
Katja J Ottmüller ◽  
Duc Dung Le ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Lamina Propria ◽  
Stromal Cells ◽  
Cd4 T Cells ◽  
Initiation Phase ◽  
Cell Responses ◽  
Alloreactive T Cells ◽  
Alloreactive T Cell

Allogeneic T cell priming is considered as an essential event determining the outcome of allogeneic hematopoietic stem cell transplantation (allo-HCT), ideally triggering anti-leukemic responses (GvL effect) or, at worst, causing life-threatening acute graft-versus-host disease (aGvHD). During acute GvHD initiation, alloreactive T cells are activated by host antigen presenting cells (APCs), rapidly expand and subsequently exert tissue damage. Recently, it was discovered that absence of host hematopoietic APCs does not prevent acute GvHD, suggesting a crucial role of non-hematopoietic APCs for priming alloreactive T cells (Toubai et al., Blood 2012, Li et al., J Immunol. 2012). Furthermore, it was even suggested that in the absence of professional APCs allogeneic CD4+ T cells can be activated in the lamina propria by MHC class II expressing myofibroblasts (Koyama et al., Nat Med 2012). As exact location and identity of host non-hematopoietic APCs triggering alloreactive T cell responses are essential to dissect the priming of GvHD-inducing vs. GvL-mediating allogeneic T cells, we investigated the role of lymph node stromal cells (LNSCs) in the initiation phase of aGvHD and their potential role as non-hematopoietic APCs. Employing allo-HCT mouse models in combination with flow cytometry and advanced microscopy techniques, we explored early alloreactive T cells activation first in a myeloablatively conditioned MHC major mismatch allo-HCT setting (FVB→B6). Under these conditions, CD4+ and CD8+ T cells activation and proliferation occurred exclusively in secondary lymphoid organs (SLOs) and not in the intestinal lamina propria early after allo-HCT within the first three days after allo-HCT. To study non-hematopoietic antigen presentation early after allo-HCT, we generated bone marrow B6.MHCIIΔ/Δ→B6.WT chimeras that lacked MHC II expression in the host hematopoietic compartment. Subsequent allo-HCT of these chimeras revealed activation and expansion of allogenic donor CD4+ T-cells exclusively in SLOs and not the lamina propria in the first three days after transplantation. Next, we generated recipient mice that selectively lacked MHCII expression either in CD11c expressing cells or under the control of the Vav1-promoter in all hematopoietic cells. In both type of recipients, allogenic donor CD4+ T-cells were activated within 3 days after allo-HCT in SLOs and no other tissues. After irradiation of B6.WT mice we observed LNSCs upregulate co-stimulatory receptors early after irradiation (24 and 72 hours), in vitro and in vivo suggesting that they may act as active non-hematopoietic APCs. Next, we performed mixed lymphocyte reactions (MLRs) of irradiated APCs derived from TgVav1-Cre+MHCIIΔ/Δmice with alloreactive CD4+ T cells from FVB mice. Here, we observed in the total absence of hematopoietic MHCII antigen presentation reduced but still pronounced activation of alloreactive CD4+ T cells. Therefore, in these transgenic allo-HCT models, hematopoietic cells in the SLOs were dispensable for alloreactive CD4+ T cell activation in the initiation phase of aGvHD. To ascertain that SLOs are the exclusive priming sites of alloreactive CD4+ T cells, we transplanted mesenteric lymph nodes (mLNs) from a B6.CD11c.DTR mice, depleted of CD11c+ cells into B6.MHCIIΔ/Δmice. After successful engraftment of donor mLNs in these mice, allo-HCT revealed these as unique priming sites in MHCII deficient hosts for alloreactive CD4+ T cells, which differentiated into CD44hiCD62Llow effector T cells that were detectable in the transplanted mLNs. Conclusively, these results indicate that specialized non-hematopoietic lymph node stromal cells prime alloreactive CD4+ T cell within the SLOs early after allo-HCT. Pinpointing the molecular pathways in these non-hematopoietic APCs within SLOs that trigger alloreactive T cell responses may prove fruitful for selective therapeutic intervention after allo-HCT. 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 T cell–specific STAT3 deficiency abrogates lupus nephritis

Lupus ◽  
2019 ◽  
Vol 28 (12) ◽  
pp. 1468-1472 ◽  
Author(s):  
N Yoshida ◽  
F He ◽  
V C Kyttaris
Keyword(s):  
T Cells ◽  
T Cell ◽  
Lupus Nephritis ◽  
Lupus Erythematosus ◽  
Cell Responses ◽  
Cytokines And Chemokines ◽  
Novel Approach ◽  
Cell Tissue ◽  
External Stimuli

Signal transducer and activator of transcription (STAT) 3 is a regulator of T-cell responses to external stimuli, such as pro-inflammatory cytokines and chemokines. We have previously shown that STAT3 is activated (phosphorylated) at high levels in systemic lupus erythematosus (SLE) T cells and mediates chemokine-induced migration and T:B cell interactions. Stattic, a small molecular STAT3 inhibitor, can partially ameliorate lupus nephritis in mice. To understand the role of STAT3 better in T-cell pathophysiology in lupus nephritis and its potential as a treatment target, we silenced its expression in T cells using a cd4-driven CRE-Flox model. We found that lupus-prone mice that do not express STAT3 in T cells did not develop lymphadenopathy, splenomegaly, or glomerulonephritis. Moreover, the production of anti-dsDNA antibodies was decreased in these mice compared to controls. To dissect the mechanism, we also used a nephrotoxic serum model of nephritis. In this model, T cell–specific silencing of STAT3 resulted in amelioration of nephrotoxic serum-induced kidney damage. Taken together, our results suggest that in mouse models of autoimmune nephritis, T cell–specific silencing of STAT3 can hamper their ability to help B cells to produce autoantibodies and induce cell tissue infiltration. We propose that STAT3 inhibition in T cells represents a novel approach in the treatment of SLE and lupus nephritis in particular.

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