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.

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.

Modulation of acute graft-versus-host disease and chimerism after adoptive transfer of in vitro-expanded invariant Vα14 natural killer T cells

2006 ◽  
Vol 106 (1) ◽  
pp. 82-90 ◽  
Author(s):  
Masaki Kuwatani ◽  
Yoshinori Ikarashi ◽  
Akira Iizuka ◽  
Chihiro Kawakami ◽  
Gary Quinn ◽  
...  
Keyword(s):  
T Cells ◽  
Natural Killer ◽  
Graft Versus Host Disease ◽  
Adoptive Transfer ◽  
Natural Killer T Cells ◽  
Host Disease ◽  
Graft Versus Host ◽  
Killer T Cells ◽  
Acute Graft

The Role of Selective Depletion of Alloreactive Donor T Cells in Graft-Versus-Host Disease after Allogeneic Bone Marrow Transplantation.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 5167-5167
Author(s):  
Yihuan Chai ◽  
Huiying Qiu ◽  
Hui Lv
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Graft Versus Host Disease ◽  
Acute Gvhd ◽  
Allogeneic Bone Marrow ◽  
Third Party ◽  
Allogeneic Bone ◽  
Host Disease ◽  
Graft Versus Host ◽  
Donor T Cells

Abstract One of the main goals in allogeneic bone marrow(BM) transplantation is the abrogation of graft-versus-host disease (GVHD) with the preservation of antileukemia and antiviral activity. The Study present a selective T cell depletion strategy based on the physical separation of the alloreactive T cells, which were identified by expression of two activation-induced antigens (CD25 and CD69). T cells from C57BL/6(H-2b) mice were first activated with BALB/c (H-2d) recipient spleen cells in a 2-day mixed-lymphocyte-culture (MLC). Following this activation, this compound is selectively depleted based on expression of two activation-induced antigens CD25 and CD69 using magnetic cell sorting. The depleted cells or the untreated cells were then rechallenged respectively in a secondary MLC, with the same stimulator cells or a third-party (DBAH-2k) or tumor- specific (SP2/0, BALB/c-origin myeloma) cells. Cells proliferation were assayed at the indicated time points(1, 2, 3, 4, 5 days). These treated cells or control-cultured cells (2.0×106) mixed with 5.0×106 BM cells from C57BL/6 were transfused respectively by the trail vain into the lethally irradiated BALB/c to observe the survival time, GVHD incidence and pathological analysis. MLC assays demonstrated that this technique led to a significant decrease in alloreactivity of donor cells(29.02~64.17%), which at the same time preserved reactivity against third party cells(49.61~75.69%)and anti-tumor cells(61.14~68.62%). The mice in the group of control-coclutured were died of acute GVHD within 24days. The 7 recipient mice in the treated group were free of acute GVHD, and 3 mice were died of acute GVHD (aGVHD) within 23 days. MACS-based ex-vivo depletion of alloreactive donor T cells based on expression of two activation-induced antigens (CD25 and CD69) could inhibit anti-host responses, by contrast, anti-SP2/O and anti-third-party responses were preserved. Cotransplantation of these selected depleted cells and BM cells could reduce aGVHD.

Adoptive Transfer Of Ex Vivo “Educated” CD4+CD25+FoxP3+ Regulatory T Cells Effectively Treats Acute Graft Versus Host Disease Preserving Graft Versus Tumor Effect

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4485-4485
Author(s):  
Antonio Pierini ◽  
Dominik Schneidawind ◽  
Mareike Florek ◽  
Maite Alvarez ◽  
Yuqiong Pan ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Tumor Cells ◽  
Graft Versus Host Disease ◽  
Adoptive Transfer ◽  
Cellular Therapy ◽  
Ex Vivo ◽  
Host Disease ◽  
Graft Versus Host

Donor derived regulatory T cells (Tregs) effectively prevent graft versus host disease (GVHD) in mouse models and in early phase clinical trials. Interleukin 2 (IL-2) therapy in patients with chronic GVHD (cGVHD) can increase Treg number and the Treg/CD4+ T cell ratio resulting in organ damage reduction and symptom relief. Less is known regarding Treg-based treatment for acute GVHD (aGVHD). In this study we evaluated the role of donor Treg cellular therapy for aGVHD treatment in well established murine models. T cell depleted bone marrow (TCD BM) from C57BL/6 mice was transplanted into lethally irradiated (8 Gy) BALB/C recipients together with 7.5x105 to 1x106/animal donor derived luc+ Tcons. Naturally occurring CD4+CD25+FoxP3+ donor type Tregs (nTregs) were purified from C57BL/6 donor mice. 2.5x105/mouse nTregs were injected at day 6 or 7 after transplant in mice that showed clear clinical signs of aGVHD and Tcon proliferation assessed by bioluminescence imaging (BLI). Survival analysis showed a favorable trend for nTreg treated mice, but the impact of this treatment was modest and not statistically significant (p 0.08). aGVHD is a disease characterized by the activation and rapid proliferation of alloreactive donor conventional T cells (Tcons) directed against host antigens, so one of the major obstacles of this approach is to overcome the large number and effector function of activated Tcons. Several studies have utilized ex vivo expansion of Tregs to increase their number with the goal of maintaining suppressive function. We developed a different strategy with the intent to “educate” Tregs to specifically suppress the reactive Tcon population. We incubated 2.5x105 donor derived Tregs with irradiated (3000 cGy) blood of aGVHD affected mice for 20 hours without further stimulation and injected the entire pool of these cells, termed educated Treg (eTregs), at day 7 or 8 after transplant and Tcon injection. Interestingly eTregs significantly improved aGVHD affected mouse survival (p = 0.0025 vs Tcons alone). BLI showed no difference between the groups (p = 0.85) because the treatment intervened after Tcon proliferation and activation was initiated. To evaluate eTreg impact on graft versus tumor (GVT) effects, we transplanted BALB/C mice with C57BL/6 TCD BM and 1x104/mouse luc+ A20 tumor cells along with 1x106/mouse donor Tcons and 2.5x105 eTregs. Mice that received TCD BM and A20 tumor cells alone died from progressive tumor growth, while mice that received Tcons died from GVHD without tumor engraftment. Further animals that received both Tcon and eTreg treatment did not have tumor engraftment demonstrating that eTregs do not impact Tcon mediated GVT effects. Further studies are ongoing to characterize the eTreg population as compared to nTreg, with respect to expression of activation markers and in functional assays. Our observations indicate that Tregs can be ex vivo educated to suppress in vivo reactive and proliferating Tcons. Moreover our data demonstrate that eTreg adoptive transfer is clinically feasible and promising. These findings may be relevant for the development of clinical grade Treg based cellular therapy for the treatment of conditions caused by immune dysregulation such as aGVHD and autoimmune diseases and for transplant tolerance induction. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Third-party umbilical cord blood–derived regulatory T cells prevent xenogenic graft-versus-host disease

Cytotherapy ◽  
2014 ◽  
Vol 16 (1) ◽  
pp. 90-100 ◽  
Author(s):  
Simrit Parmar ◽  
Xiaoying Liu ◽  
Shawndeep S. Tung ◽  
Simon N. Robinson ◽  
Gabriel Rodriguez ◽  
...  
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Cord Blood ◽  
Umbilical Cord ◽  
Graft Versus Host Disease ◽  
Umbilical Cord Blood ◽  
Third Party ◽  
Host Disease ◽  
Graft Versus Host

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.

Adoptive transfer of allogeneic regulatory T cells into patients with chronic graft-versus-host disease

Cytotherapy ◽  
2015 ◽  
Vol 17 (4) ◽  
pp. 473-486 ◽  
Author(s):  
Anke Theil ◽  
Sebastian Tuve ◽  
Uta Oelschlägel ◽  
Anja Maiwald ◽  
Diana Döhler ◽  
...  
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Graft Versus Host Disease ◽  
Adoptive Transfer ◽  
Host Disease ◽  
Graft Versus Host

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.

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