scholarly journals Invariant Natural Killer T Cell Subsets Have Diverse Functions: iNKT2 and iNKT17 Protect from Graft-Versus-Host-Disease, Whereas iNKT1 Have Antitumor Potential

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 475-475
Author(s):  
Kristina Maas-Bauer ◽  
Federico Simonetta ◽  
Toshihito Hirai ◽  
Arielle Wenokur ◽  
Furqan Fazal ◽  
...  
Keyword(s):  
T Cells ◽  
Natural Killer ◽  
Board Of Directors ◽  
Graft Versus Host Disease ◽  
Survival Benefit ◽  
Inkt Cells ◽  
Advisory Committees ◽  
Host Disease ◽  
Graft Versus Host ◽  
Invariant Natural Killer

Abstract Invariant natural killer T (iNKT) cells are an interesting subpopulation of T cells that can potently inhibit graft-versus-host-disease (GVHD) through the production of Interleukin 4 (IL-4), while also carrying anti-tumor potential (Leveson-Gower et al. Blood. 2011;117:3220-9; Schneidawind et al. Blood. 2014;124:3320-8). Murine iNKT cells differentiate during thymic development into three distinct sublineages, named according to the classification of conventional T cells: Th1-like iNKT (iNKT1) cells, Th2-like iNKT (iNKT2) cells, and Th-17 like iNKT (iNKT17) cells (Brennan et al. Nat Rev Immunol. 2013; 13:101-1). In this study we investigated the immune regulatory and anti-tumor potential of iNKT1, iNKT2 and iNKT17 cell subsets. Thymic iNKT1 cells, iNKT2 cells and iNKT17 cells were isolated from 8-10 week-old FVB/NJ mice by flow cytometry based on PBS-57-CD1d-Tetramer and a combination of cell surface molecules (iNKT1: ICOS- PD1- CD27+ CD24-; iNKT2: ICOS+ PD1+ CD27+ CD4+ CD24-; iNKT17: ICOS+ PD1+ CD4- CD27- CD24-) and purity was confirmed by intra-nuclear staining for the transcription factors PLZF and RORγT. RNA sequencing analysis determined that iNKT1 cells were the main subset expressing proinflammatory and cytotoxic genes, such as Interferon gamma (IFN-γ), Fas Ligand (FasL), Perforin, and Granzyme B (Gzmb), whereas IL-4 was expressed by iNKT2 cells and, at a lesser extent, by iNKT17 cells. To assess the immuno-regulatory potential of the three iNKT sublineages, we employed a murine major histocompatibility complex (MHC)-mismatched bone marrow transplantation model. BALB/c (H-2Kd) recipients were lethally irradiated with 8.8 Gy; on the same day, 4 x 106 TCD-BM cells and 1 x 106 conventional CD4 and CD8 T cells (Tcon) from FVB/NJ (H-2kq) mice were injected intravenously. Additionally, 5 x 104 purified iNKT1, iNKT2 or iNKT17 cell subsets from FVB donors were injected. A significant survival benefit was observed when iNKT2 (p=0.017) and iNKT17 (p=0.033) cells were adoptively transferred compared to mice that only received TCD-BM and Tcon, whereas there was no survival benefit in the group that received iNKT1 cells. In addition, body weight was improved in mice that received iNKT2 (day +41: p=0.009, day +49: p=0.005 and day +59: p= 0.005) or iNKT17 (day +59: p= 0.006) compared to mice that received iNKT1 cells. Clinical GVHD scores were also improved in mice that received iNKT2 (day +41: p= 0.012, day +51: p= 0.005) or iNKT17 (day +28: p=0.05, day +51: p=0.007) compared to mice that received iNKT1 cells. Interestingly, we found that even 1 x104 iNKT2 (p= 0.008) and iNKT17 (p= 0.04) significantly suppressed GVHD. As iNKT1, iNKT2 and iNKT17 have a very different gene expression profile, we tested the ability of the sublineages to kill a B-cell lymphoma cell line transduced to express high levels of CD1d (A20-CD1d) in vitro and found that iNKT1 cells killed A20-CD1d cells significantly better than iNKT2 (p=0.006) or iNKT17 (p=0.0001) cells. These findings are in line with the sequencing data mentioned above, showing that iNKT1 cells express a more inflammatory phenotype. In summary, we demonstrate here that only iNKT2 and iNKT17 cells protect from GVHD, whereas iNKT1 cells have cytotoxic function. To our knowledge, this is the first study to show functional differences between the iNKT sublineages, suggesting that iNKT1, iNKT2 and iNKT17 cells have diverse functions. Therefore, these data provide new biological insights, which will be useful for developing iNKT cell-based cell therapy. Disclosures Chang: Spring Discovery: Membership on an entity's Board of Directors or advisory committees; Epinomics and Accent Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Third Party Invariant Natural Killer T Cells Protect from Graft-Versus-Host Disease Lethality through Expansion of Donor CD4+FoxP3+ Regulatory T Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3825-3825
Author(s):  
Dominik Schneidawind ◽  
Jeanette Baker ◽  
Corina Buechele ◽  
Everett H. Meyer ◽  
Robert S. Negrin
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Natural Killer ◽  
Graft Versus Host Disease ◽  
Adoptive Transfer ◽  
Third Party ◽  
Inkt Cells ◽  
Graft Versus Host ◽  
Donor T Cells ◽  
Invariant Natural Killer

Abstract Graft-versus-host disease (GVHD) is driven by extensive activation and proliferation of alloreactive donor T cells causing significant morbidity and mortality following allogeneic hematopoietic cell transplantation (HCT). Invariant natural killer T (iNKT) cells are potent regulators of immune responses in both humans (TCRα Vα24-Jα18) and mice (TCRα Vα14-Jα18). As the iNKT cell receptor and the glycolipid-presenting molecule CD1d interaction is highly conserved, we explored the role of adoptively transferred third party CD4+ iNKT cells in a murine model of allogeneic HCT. BALB/c (H-2Kd) recipient mice were irradiated with 8 Gy and transplanted with T cell-depleted bone marrow together with 1x106 CD4+/CD8+ T cells (Tcon) from C57BL/6 (H-2Kb) donor mice. Adoptive transfer of purified (>95%) 5x104 CD4+ iNKT cells from FVB/N (H-2Kq) third party mice resulted in a significant survival benefit (p<0.001) while retaining Tcon mediated graft-versus-tumor (GVT) effects against A20 lymphoma cells (p=0.002). Consistently, weight and GVHD scores improved in mice that received a single injection of third party CD4+ iNKT cells as compared to animals that received Tcon alone. Notably, CD4+ iNKT cells from third party mice were as protective as CD4+ iNKT cells from donor mice (p=0.50). Signal intensity deriving from expanding luciferase expressing alloreactive Tcon was significantly lower in animals treated with third party CD4+ iNKT cells (p=0.003). Interestingly, inhibition of Tcon proliferation was similar to animals that received CD4+ iNKT cells from donor mice (p=0.90). In addition, adoptive transfer of third party CD4+ iNKT cells promoted a Th2-biased cytokine response of alloreactive donor T cells. Although we found that third party CD4+ iNKT cells were rejected by day +10 after allogeneic HCT, adoptive transfer of these cells resulted in a robust expansion of luciferase expressing donor CD4+FoxP3+ regulatory T cells (Treg) as measured by bioluminescence imaging (p=0.006). Using FoxP3DTR C57BL/6 donor mice, depletion of Treg from the graft abrogated both donor Treg expansion and protection from GVHD lethality through third party CD4+ iNKT cells. We conclude that low numbers of highly purified and adoptively transferred third party CD4+ iNKT cells protect from lethal GVHD through activation and expansion of donor Treg with retained GVT effects. Despite the fact that iNKT cells are a rare cell population, the in vivo activity of small numbers of cells and feasibility of in vitro expansion provide the basis for clinical translation. Disclosures No relevant conflicts of interest to declare.

The Novel Combination of Sirolimus and Bortezomib Prevents Graft-Versus-Host Disease but Mantains the Graft-Versus-Leukemia Effect After Allogeneic Transplantation.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3738-3738
Author(s):  
Teresa Caballero-Velazquez ◽  
Luis Ignacio Sanchez-Abarca ◽  
Belen Blanco ◽  
Carmen Herrero ◽  
Silvia Gutierrez-cosio ◽  
...  
Keyword(s):  
T Cells ◽  
Synergistic Effect ◽  
Board Of Directors ◽  
Graft Versus Host Disease ◽  
Allogeneic Transplantation ◽  
Advisory Committees ◽  
Host Disease ◽  
Graft Versus Host ◽  
Gvhd Prophylaxis

Abstract Abstract 3738 Graft-versus-host disease (GVHD) represents a major challenge and the main cause of morbidity and mortality after allogeneic transplantation. Using the standard GVHD prophylaxis based on a calcineurin inhibitor plus methotrexate (MTX). The incidence of acute GVHD is in the range of 30–60%, so that new strategies are required in order to decrease GVHD without hampering GVL. Sirolimus, an mTOR inhibitor, allows to decrease the risk of GVHD and increases the number of Treg after transplantation. Unfortunately, its use may increase the risk of microangiopathy and, moreover, its combination with calcineurin inhibitors blocks the development of Treg. Bortezomib is a proteosome inhibitor and blocks the nuclear translocation and activation of NF-kB. It induces depletion of alloreactive T and allows the expansion of T-cells with suppressive properties. Accordingly, both drugs could favour the development of a tolerogeneic immune response after transplantation. In the current study we have analyzed the synergistic effect of sirolimus together with bortezomib. Bortezomib 100 nM plus sirolimus 5nM synergistically inhibited T-cell activation as assessed by the expression of CD25, production of IFNg and expression of CD40L as well as proliferation assessed as expression of PKH. Remain vibility, these effects could not be attributed to a decreased viability of T-cells, as assessed by 7-AAD, at the concentrations evaluated. As compared to each drug alone, the combination significantly decreased the production of Th1 cytokines (IFNg, IL-2 and TNF) while regarding TH2 cytokines, only IL-6 significantly decreased upon combining both drugs. Concerning the mechanisms involved in this synergistic effect, the combination of both drugs resulted in an inhibition of the Akt and Erk ½ phophorylation, thus indicating that sirolimus inhibit pathways which could allow T-cells to escape from the effect of bortezomib at the doses used in the current experiment. In order to confirm in vivo the synergistic effect of sirolimus and bortezomib, a GVHD mouse model (C57/BL6-Balb/c) was carried out. Mice receiving both drugs (Bortezomib 1μg/day intravenous on days 0, +1, +2 postransplantat and sirolimus 0.25mg/Kg intra-peritoneal on days 0 to 12) has a significantly lower incidence of GVHD and longer survival as compared to each drug alone. We also wanted to evaluate whether the immunosuppressive effect of the combination was unspecific or, by contrast, it allowed induce specific immune tolerance against host but maintaining the immune response against other antigens. For this purpose a haematopoietic cells of Balb/c mice which were C57/BL6 complete chimeras were infused to NOD-SCID mice. While none of the donors had developed GVHD after transplantation plus sirolimus and bortezomib postransplant, the NOD-SCID mice succumbed due to GVHD. Furthermore, we infused WEHI cells to BALB/c after total body irradiation and we observed that, while all BALB/c mice receiving WEHI plus C57BL/6 donor BM cells died due to leukemic infiltration, none of those receiving WEHI cells plus C57BL/6 donor BM cells plus splenocytes (and GVHD prophylaxis with sirolimus and bortezomib) did develop leukemic infiltration, thus confirming that, using this approach, we were able to separate GVHD and GVL effect. In conclusion, the current study shows a potent synergistic effect between sirolimus and bortezomib in vitro and in vivo which prevent GVHD while maintaining GVL. Disclosures: Cañizo: CELGENE: Membership on an entity's Board of Directors or advisory committees. San Miguel:JANSSEN-CILAG: Membership on an entity's Board of Directors or advisory committees; CELGENE: Membership on an entity's Board of Directors or advisory committees; NOVARTIS: Membership on an entity's Board of Directors or advisory committees; MILLENNIUM: Membership on an entity's Board of Directors or advisory committees. Off Label Use: sirolimus and bortezomib are not approved for use in prophylaxis of graft versus host disease.

CD4+ Invariant Natural Killer T Cells Require NKG2D To Protect From Lethal Acute Graft-Versus-Host Disease

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 898-898
Author(s):  
Dominik Schneidawind ◽  
Antonio Pierini ◽  
Maite Alvarez ◽  
Yuqiong Pan ◽  
Jeanette Baker ◽  
...  
Keyword(s):  
Lymph Nodes ◽  
Natural Killer ◽  
Cell Population ◽  
Graft Versus Host Disease ◽  
Acute Gvhd ◽  
Inkt Cells ◽  
Host Disease ◽  
Graft Versus Host ◽  
Invariant Natural Killer

Abstract Invariant Natural Killer T (iNKT) cells are a rare cell population in humans (TCRα Vα24-Jα18) and mice (TCRα Vα14-Jα18) that are characterized by a rapid release of immunoregulatory cytokines upon stimulation. The tolerogenic impact of host iNKT cells on graft-versus-host disease (GVHD) after conditioning with total lymphoid irradiation and anti-thymocyte globulin (TLI/ATG) has been shown previously (Lan et al., BBMT 2003). Moreover, we recently described a subset of CD49b+ NKT cells overlapping with iNKT cells and providing protection from acute GVHD mainly via an IL-4-dependent mechanism (Leveson-Gower et al., Blood 2011). Here, we investigated the role of highly purified adoptively transferred donor-derived CD4+ iNKT cells. Balb/c recipient mice were transplanted with T cell-depleted bone marrow together with 1x106 CD4/CD8 T lymphocytes (Tcon) from C57Bl/6 donor mice after irradiation with 8 Gy. Mice co-injected with as low as 5x104 freshly isolated and highly purified (>99%) CD4+ iNKT cells showed a significant survival benefit compared to mice receiving Tcon alone (p=.0015). Consistently, weight and GVHD score improved in mice that received CD4+ iNKT cells. Signal intensity derived from expanding luciferase expressing alloreactive Tcon was significantly lower in animals treated with CD4+ iNKT cells demonstrating inhibition of proliferation of alloreactive Tcon through CD4+ iNKT cells (p<.0001). In vivo CFSE proliferation assay confirmed decreased Tcon proliferation in peripheral lymph nodes (p<.0001), mesenteric lymph nodes (p=.0277) and spleen (p=.0005). CD4+ iNKT cells showed a Th2-biased cytokine profile with high levels of IL-4 and IL-13 in the presence of alloreactive Tcon challenged with irradiated allogeneic stimulators. Co-injection of CD4+ iNKT cells that were expanded 5-fold in vitro with α-GalCer and IL-2 had the same protective effect from lethal acute GVHD compared to freshly isolated CD4+ iNKT cells (p=.7987). Interestingly, CD4+ iNKT cells derived from NKG2D-/- animals were significantly less effective in preventing acute GVHD lethality (WT vs. NKG2D-/-p=.0027). In conclusion, low numbers of highly purified freshly isolated and cultured CD4+ iNKT cells protect from lethal acute GVHD in mice and require NKG2D. Despite the fact that iNKT cells are a rare cell population, the feasibility of in vitro expansion with retained functionality of CD4+ iNKT cells provide the basis for clinical translation. EM and RN contributed equally. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Invariant Natural Killer T Cell Subsets Have Diverse Graft-Versus-Host-Disease-Preventing and Anti-Tumor Effects

Blood ◽  
2021 ◽  
Author(s):  
Kristina Maas-Bauer ◽  
Juliane K. Lohmeyer ◽  
Toshihito Hirai ◽  
Teresa Lopes Ramos ◽  
Furqan Muhammad Fazal ◽  
...  
Keyword(s):  
T Cell ◽  
Antitumor Activity ◽  
Natural Killer ◽  
Graft Versus Host Disease ◽  
Inkt Cells ◽  
Host Disease ◽  
Graft Versus Host ◽  
Invariant Natural Killer

Invariant Natural Killer T (iNKT) cells are a T cell subset with potent immunomodulatory properties. Experimental evidence in mice and observational studies in humans indicate that iNKT cells have antitumor potential as well as the ability to suppress acute and chronic Graft-versus-Host-Disease (GvHD). Murine iNKT cells differentiate during thymic development into iNKT1, iNKT2 and iNKT17 sublineages, which differ transcriptomically and epigenomically, and have subset-specific developmental requirements. Whether distinct iNKT sublineages also differ in their antitumor effect and their ability to suppress GvHD is currently unknown. In this work, we generated highly purified murine iNKT-sublineages, characterized their transcriptomic and epigenomic landscape, and assessed specific functions. We demonstrate that iNKT2 and iNKT17, but not iNKT1 cells, efficiently suppress T cell activation in vitro and mitigate murine acute GvHD in vivo. Conversely, we show that iNKT1 cells display the highest antitumor activity against murine B-cell lymphoma cells both in vitro and in vivo. Thus, we demonstrate for the first time that iNKT sublineages have distinct and different functions, with iNKT1 cells having the highest antitumor activity and iNKT2 and iNKT17 cells having immune-regulatory properties. These results have important implications for the translation of iNKT cell therapies to the clinic for cancer immunotherapy as well as for GvHD prevention and treatment.

scholarly journals Early posttransplantation donor-derived invariant natural killer T-cell recovery predicts the occurrence of acute graft-versus-host disease and overall survival

Blood ◽  
2012 ◽  
Vol 120 (10) ◽  
pp. 2144-2154 ◽  
Author(s):  
Marie-Thérèse Rubio ◽  
Lucia Moreira-Teixeira ◽  
Emmanuel Bachy ◽  
Marie Bouillié ◽  
Pierre Milpied ◽  
...  
Keyword(s):  
Overall Survival ◽  
Natural Killer ◽  
Graft Versus Host Disease ◽  
Cell Recovery ◽  
Inkt Cells ◽  
Hematopoietic Stem ◽  
Host Disease ◽  
Graft Versus Host ◽  
Invariant Natural Killer ◽  
Natural Killer T

Abstract Invariant natural killer T (iNKT) cells can experimentally dissociate GVL from graft-versus-host-disease (GVHD). Their role in human conventional allogeneic hematopoietic stem cell transplantation (HSCT) is unknown. Here, we analyzed the post-HSCT recovery of iNKT cells in 71 adult allografted patients. Results were compared with conventional T- and NK-cell recovery and correlated to the occurrence of GVHD, relapse, and survival. We observed that posttransplantation iNKT cells, likely of donor origin, recovered independently of T and NK cells in the first 90 days after HSCT and reached greater levels in recipient younger than 45 years (P = .003) and after a reduced-intensity conditioning regimen (P = .03). Low posttransplantation iNKT/T ratios (ie, < 10−3) were an independent factor associated with the occurrence of acute GVHD (aGVHD; P = .001). Inversely, reaching iNKT/T ratios > 10−3 before day 90 was associated with reduced nonrelapse mortality (P = .009) without increased risk of relapse and appeared as an independent predictive factor of an improved overall survival (P = .028). Furthermore, an iNKT/T ratio on day 15 > 0.58 × 10−3 was associated with a 94% risk reduction of aGVHD. These findings provide a proof of concept that early postallogeneic HSCT iNKT cell recovery can predict the occurrence of aGVHD and an improved overall survival.

scholarly journals Murine Donor 1928z CAR T Cells Exert Potent Graft-Versus-Lymphoma Activity without Graft-Versus-Host-Disease

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 653-653
Author(s):  
Melody Smith ◽  
Arnab Ghosh ◽  
Scott James ◽  
Marco L Davila ◽  
Enrico Velardi ◽  
...  
Keyword(s):  
T Cells ◽  
Board Of Directors ◽  
Graft Versus Host Disease ◽  
Adoptive Transfer ◽  
Transcriptional Analysis ◽  
Advisory Committees ◽  
Host Disease ◽  
Graft Versus Host ◽  
Car T Cells ◽  
Car T

Abstract Chimeric antigen receptors (CARs) promote tumor cell target recognition of adoptively transferred T cellsand clinical studies have shown that autologous T cells transduced with CARs targeting CD19, expressed on normal B cells and several B cell malignancies, are effective against CD19+ malignancies. The potential of donor-derived CD19-CAR T cells to treat relapse after allogeneic hematopoietic stem cell transplantation (alloHSCT) has been relatively unexplored due to concerns for graft-versus-host disease (GVHD) and other toxicities. However, recent clinical trials of donor-derived CD19-CAR T cells in allo-HSCT have noted a surprisingly low incidence of GVHD. We studied the GVHD and anti-tumor potential of donor-derived mouse CD19-specific CAR 1928z (m1928z) T cells in preclinical allo-HSCT and lymphoma models. We constructed a panel of retroviral vectors targeting mouse CD19 (Figure 1A) to test various aspects of CAR stimulation and co-stimulation and generated T cells expressing these constructs. We then used these constructs in mouse allo-HSCT models comprising BALB/c recipients and B6 donors (Figure 1B). A20 lymphoma-inoculated BALB/c recipients of B6 BM treated with donor B6 m1928z T cells had significantly higher tumor-free survival and improved survival compared to m19delta treated recipients (Figure 1C, P < 0.0001). Adoptive transfer of B6 m1928z T cells caused significantly less GVHD compared to B6 m19delta T cells (P < 0.001) in BALB/c recipients of B6 BM confirming that m1928z T cells cause less GVHD. We explored the requirement of CAR signaling for attenuated GVHD using hum1928z (P < 0.001) (anti-human CD19-CAR, non reactive in mice) T cells and noted significantly worse survival and GVHD scores in mice as compared to with m1928z indicating that CAR signaling was necessary for this protection (Figure 1D). We also examined the importance of co-stimulation and noted increased GVHD mediated by m19z T cells (P < 0.05) (CAR with no co-stimulation) (Figure 1E) and m19BBz (P < 0.01) (CAR with 4-1BB co-stimulation) (Figure 1F) indicating that CD28 co-stimulation was also vital for attenuated GVHD. To study the effect of concurrent TCR and CAR signaling in vitro, we used T cells engineered to co-express 1928z CAR and OT-1 TCR in a cold-target inhibition killing assay and noted that activation of the T cells through 1928z can impair lytic activity mediated by TCR. To elucidate the consequence of concurrent TCR and CAR activation, we studied the TCR signaling pathway and found increased phosphorylated PKCa, pERK1/2, pS6, pSTAT1, pSTAT3, and pSTAT5 in donor m1928z T cells compared to m19delta T cells, indicating a hyperactivated state. In order to further study the effect of concurrent stimulation of alloreactive TCR and m1928z CAR, we utilized B6 ABM-RAG1 knockout (ABM) T cells, which express monoclonal TCR specific to I-Abm12 expressed on B6.C-H2bm12 (BM12) mice. In accordance with our previous findings, we observed significantly less GVHD in allo-HSCT BM12 recipients of ABM m1928z T cells versus m19delta T cells (P < 0.0001). We performed global transcriptional analysis of ABM m1928z T cells and m19delta harvested from adoptively transferred BM12 recipients of B6 BM and differential upregulation of Bim, Bid, FasL and Apaf1 in m1928z T cells. Phenotypic analysis of these cells also showed increased PD-1 and Fas expression, consistent with post-stimulatory programmed cell death In conclusion, we show that donor m1928z T cells mediate strong GVT effects with decreased GVHD. Following adoptive transfer, alloreactive m1928z T cells (but not m19BBz) undergo progressive loss of effector function and proliferative potential, and eventual deletion, which significantly decreases graft-versus-host activity. The non-alloreactive m1928z T cells present in bulk donor T cell populations retain significant anti-lymphoma activity. Our findings support the administration of donor 1928z T cells to prevent relapse of CD19+ malignancies after allo-HSCT and provide mechanistic insight into recent clinical trial results demonstrating that allogeneic CD19-specific 1928z CAR T cells promote anti-lymphoma activity while causing minimal GVHD. Figure 1. Figure 1. Disclosures Davila: Adaptive Biotechnologies: Membership on an entity's Board of Directors or advisory committees; Celyad: Consultancy; Precision Biosciences: Membership on an entity's Board of Directors or advisory committees; Kite Pharma: Membership on an entity's Board of Directors or advisory committees; Servier: Membership on an entity's Board of Directors or advisory committees. Sadelain:Juno Therapeutics: Consultancy, Equity Ownership, Patents & Royalties.

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