iNKT Cell Numbers Research Articles

Abstract 3769: Stem cell-engineered invariant natural killer T cells for cancer therapy

Abstract Invariant natural killer T (iNKT) cells comprise a small population of αβ T lymphocytes. They bridge the innate and adaptive immune systems and mediate strong and rapid responses to many diseases, including cancer, infections, allergies and autoimmunity. However, the study of iNKT cell biology and the therapeutic applications of these cells are greatly limited by their small numbers in vivo (~0.01-1% in mouse and human blood). Here, we report a new method to generate large numbers of iNKT cells in mice through T cell receptor (TCR) gene engineering of hematopoietic stem cells (HSCs). We showed that iNKT TCR-engineered HSCs could generate a clonal population of iNKT cells. These HSC-engineered iNKT cells displayed the typical iNKT cell phenotype and functionality. They followed a two-stage developmental path, first in thymus and then in the periphery, resembling that of endogenous iNKT cells. When tested in a mouse melanoma lung metastasis model, the HSC-engineered iNKT cells effectively protected mice from tumor metastasis. This method provides a powerful and high-throughput tool to investigate the in vivo development and functionality of clonal iNKT cells in mice. More importantly, this method takes advantage of the self-renewal and longevity of HSCs to generate a long-term supply of engineered iNKT cells, thus opening up a new avenue for iNKT cell-based immunotherapy. Citation Format: Lili Yang, Drake Smith, Siyuan Liu, Sunjong Ji, Bo Liu. Stem cell-engineered invariant natural killer T cells for cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3769. doi:10.1158/1538-7445.AM2017-3769

Histamine receptor 2 modifies iNKT cell activity within the inflamed lung.

Histamine is a key immunoregulatory mediator and can dampen proinflammatory responses via activation of histamine receptor 2 (H2 R). The aim of this study was to determine the role of H2 R in modulating lung inflammatory responses. H2 R was blocked using famotidine or activated using dimaprit in both the ovalbumin (OVA) and house dust mite extract (HDM) murine models of respiratory inflammation. H2 R-deficient animals and CD1d/H2 R-deficient animals were utilized to examine the CD1d presentation of lipid antigens (αGalCer or OCH) to invariant natural killer T (iNKT) cells. Famotidine treatment resulted in more severe airway disease in the OVA model, while dimaprit treatment significantly reduced disease severity. Both OVA and HDM-induced airway diseases were more severe in H2 R-deficient animals. Flow cytometric analysis of lung tissue from H2 R-deficient animals revealed increased numbers of CD1d+ dendritic cells and increased numbers of iNKT cells. In vitro, αGalCer-stimulated iNKT cells from H2 R-deficient mice secreted higher levels of IL-4, IL-5, and GM-CSF. In vivo, αGalCer or OCH administration to the lung resulted in enhanced mucus secretion, inflammatory cell recruitment, and cytokine production in H2 R-deficient or famotidine-treated animals, while dimaprit dampened the lung iNKT cell response to αGalCer. Removal of iNKT cells in H2 R-deficient (CD1d-/- H2 R-/- ) animals normalized the lung response to HDM. The deliberate activation of H2 R, or its downstream signaling molecules, may represent a novel therapeutic target for chronic lung inflammatory diseases, especially when CD1d-mediated presentation of lipid antigens to iNKT cells is contributing to the pathology.

MiR150 over-expression impairs iNKT cell development, maturation, and function

Abstract CD1d-restricted invariant natural killer T (iNKT) cells play an important role in the regulation of diverse immune responses. MicroRNAs have been shown to be critically involved in iNKT cell development, maturation, and function, but the roles of specific miRNAs in it remain not completely understood. We had previously shown the dynamic upregulation of miR-150 during thymus iNKT cell maturation and identified the interrupted iNKT final stage maturation and elevated IFNγ production in miR-150KO mice. To determine the requirement of dynamic expressional changes of miR150 during iNKT cell development, we generated thymus-specific miR150 knock in mice (miR-150KI). MiR-150 over expression resulted in reduced overall iNKT cell number in both thymus and periphery, which could be related to increased apoptosis. MiR-150KI thymus iNKT cells showed the blockade of iNKT maturation at multiple maturation transition points. Splenic iNKT with miR-150KI displayed interrupted maturation and reduced cytokine production, including IFNγ, IL-4, and TNFα, upon in vitro stimulation. Furthermore, Bone marrow chimera study confirmed the effect of miR-150KI on iNKT cell development, maturation, and function are cell autonomous. Altogether, our results indicated that the tight control of miR150 expression is required in iNKT cell development and function. Detailed molecular mechanisms of miR-150 mediated iNKT cell regulation in different developmental stages and activation status are currently under investigation.

Distribution of invariant natural killer T cells and dendritic cells in late pre-term birth without acute chorioamnionitis.

Acute chorioamnionitis (aCAM) is an important cause of pre-term birth. However, little is known about the pathogenesis of late pre-term birth without aCAM that was the most common category of pre-term birth. Here we analyze the kinetics of immune cells obtained from the decidua of women with late pre-term births with and without aCAM. Deciduas were obtained from women who underwent labor with late pre-term birth without aCAM (PB-n/aCAM) or with aCAM (PB-w/aCAM). The population of DEC-205+ dendritic cells (DCs), macrophages, invariant natural killer T (iNKT) cells, NK cells, CD8+ T cells, and CD4+ T cells were analyzed by flow cytometry. The number of iNKT cells was higher in the decidua obtained from women with PB-n/aCAM than PB-w/aCAM. DEC-205+ DCs obtained from women with PB-n/aCAM preferentially induced iNKT cell proliferation. iNKT cell accumulation with DEC-205+ DCs in PB-n/aCAM suggests that iNKT cells contribute to the onset of PB-n/aCAM.

MTORC2 regulates multiple aspects of NKT-cell development and function.

Invariant NKT (iNKT) cells bridge innate and adaptive immunity by rapidly secreting cytokines and lysing targets following TCR recognition of lipid antigens. Based on their ability to secrete IFN-γ, IL-4 and IL-17A, iNKT-cells are classified as NKT-1, NKT-2, and NKT-17 subsets, respectively. The molecular pathways regulating iNKT-cell fate are not fully defined. Recent studies implicate Rictor, a required component of mTORC2, in the development of select iNKT-cell subsets, however these reports are conflicting. To resolve these questions, we used Rictorfl/fl CD4cre+ mice and found that Rictor is required for NKT-17 cell development and normal iNKT-cell cytolytic function. Conversely, Rictor is not absolutely required for IL-4 and IFN-γ production as peripheral iNKT-cells make copious amounts of these cytokines. Overall iNKT-cell numbers are dramatically reduced in the absence of Rictor. We provide data indicating Rictor regulates cell survival as well as proliferation of developing and mature iNKT-cells. Thus, mTORC2 regulates multiple aspects of iNKT-cell development and function.

Deletion of CD1d in Adipocytes Aggravates Adipose Tissue Inflammation and Insulin Resistance in Obesity.

Adipose tissue inflammation is an important factor in obesity that promotes insulin resistance. Among various cell types in adipose tissue, immune cells actively regulate inflammatory responses and affect whole-body energy metabolism. In particular, invariant natural killer T (iNKT) cells contribute to mitigating dysregulation of systemic energy homeostasis by counteracting obesity-induced inflammation in adipose tissue. However, the molecular mechanisms by which adipose iNKT cells become activated and mediate anti-inflammatory roles in obese adipose tissue have not been thoroughly understood yet. In the current study, we demonstrate that adipocyte CD1d plays a key role in the stimulation of adipose iNKT cells, leading to anti-inflammatory responses in high-fat diet (HFD)-fed mice. Accordingly, adipocyte-specific CD1d-knockout (CD1dADKO) mice showed reduced numbers of iNKT cells in adipose tissues and decreased responses to α-galactosylceramide-induced iNKT cell activation. Additionally, HFD-fed CD1dADKO mice revealed reduced interleukin-4 expression in adipose iNKT cells and aggravated adipose tissue inflammation and insulin resistance. Collectively, these data suggest that adipocytes could selectively stimulate adipose iNKT cells to mediate anti-inflammatory responses and attenuate excess proinflammatory responses in obese adipose tissue.

Soluble γc cytokine receptor suppresses IL-15 signaling and impairs iNKT cell development in the thymus.

The soluble γc protein (sγc) is a naturally occurring splice isoform of the γc cytokine receptor that is produced by activated T cells and inhibits γc cytokine signaling. Here we show that sγc expression is also highly upregulated in immature CD4+CD8+ thymocytes but then downregulated in mature thymocytes. These results indicate a developmentally controlled mechanism for sγc expression and suggest a potential role for sγc in regulating T cell development in the thymus. Indeed, sγc overexpression resulted in significantly reduced thymocyte numbers and diminished expansion of immature thymocytes, concordant to its role in suppressing signaling by IL-7, a critical γc cytokine in early thymopoiesis. Notably, sγc overexpression also impaired generation of iNKT cells, resulting in reduced iNKT cell percentages and numbers in the thymus. iNKT cell development requires IL-15, and we found that sγc interfered with IL-15 signaling to suppress iNKT cell generation in the thymus. Thus, sγc represents a new mechanism to control cytokine availability during T cell development that constrains mature T cell production and specifically iNKT cell generation in the thymus.

Role of invariant NKT cells in lipopolysaccharide-induced lethal shock during encephalomyocarditis virus infection

Viral infections can give rise to secondary bacterial infections. In the present study, we examined the role of invariant natural killer T (iNKT) cells in lipopolysaccharide (LPS)-induced lethal shock during encephalomyocarditis virus (EMCV) infection. Wild-type (WT) mice and Jα18 gene knockout (Jα18 KO) mice were inoculated with EMCV, 5days prior to challenging with LPS. The survival rate of Jα18 KO mice subjected to EMCV and LPS was significantly higher than that of WT mice. TNF-α and nitric oxide (NO) production were increased in WT mice, than that in Jα18 KO mice, after the administration of EMCV and LPS. EMCV infection increased the number of iNKT cells and IFN-γ production by iNKT cells in WT mice. Moreover, EMCV infection enhanced the expression of Toll-like receptor 4 (TLR4) in the lung and spleen. IFN-γ also increased the expression of TLR4 in splenocytes. These findings indicated that EMCV infection activated iNKT cells, and IFN-γ secreted from the iNKT cells up-regulated the expression of TLR4 in various tissues. As a result, EMCV-infected mice were susceptible to LPS and easily developed the lethal shock. In conclusion, iNKT cells were involved in the development of LPS-induced lethal shock during EMCV infection.

Infiltration of invariant natural killer T cells occur and accelerate brain infarction in permanent ischemic stroke in mice

Invariant natural killer T (iNKT) cells are a unique subset of T cells that have been implicated in inflammation, atopy, autoimmunity, infections, and cancer. Although iNKT cells have been extensively studied over the past decade, its role in the pathogenesis of ischemic brain injury is still largely unknown. In our study, we determined whether iNKT cells infiltration occur in a mouse model of permanent cerebral ischemia. C57BL6/J male mice were treated with either alpha-galactosylceramide (α-GalCer) or vehicle control before undergoing permanent middle cerebral artery occlusion (pMCAO). α-GalCer, a glycolipid antigen, specifically activates iNKT cells by a CD1d-restricted mechanism. Using flow cytometry, 10,000 leukocytes (CD45 high cells) from the ischemic hemisphere and peripheral blood respectively were analyzed to determine the number of NK1.1+CD3+ cells at 3, 12, 24 and 48h post-pMCAO. Cerebral infarct size, brain edema and morphological characteristics were measured at the stipulated time points by 2,3,5-triphenyltetrazolium chloride (TTC) staining, weighing, and H&E staining. The levels of IFN-γ and TNF-α in brain tissue and serum were assessed by immunohistochemistry and ELISA respectively. We found that the number of iNKT cells started increasing from 12h (PB sample) and 24h (ischemic hemisphere sample) respectively in the vehicle treated group. iNKT cells infiltration occurred at an earlier time-point compared in the α-GalCer treated group (T=3H vs T=12H in PB sample; T=12H vs T=24H in ischemic hemisphere sample). Brain water content at 12h and 24h was significantly higher in pMCAO+α-GalCer mice compared to pMCAO+vehicle mice which was in turn higher than mice that underwent sham surgery. Aggravated morphological abnormalities in HE-stained neurons and significantly increased neurons with pyknotic nuclei and cavitation in the ischemic region were observed at 24h in the pMCAO+α-GalCer and pMCAO+vehicle groups. Cerebral infarct volume, neurological deficit Scores and brain edema were significantly increased at 24h in the pMCAO+α-GalCer group compared to pMCAO+vehicle group. In the pMCAO+vehicle group, the serum concentrations of TNF-α and IFN-γ were increased at 12h and 24h post-pMCAO, and remained elevated up to 48h. In mice treated with pMCAO+α-GalCer, TNF-α and IFN-γ were both increased at 12h post-pMCAO, and remained elevated up to 48h. Immunohistochemistry showed that protein expression of TNF-α and IFN-γ in brain tissues was higher in α-GalCer-treated mice. Our results demonstrate that within 48h of focal permanent cerebral ischemia, iNKT cells infiltrate into the brain and contribute to brain infarction.

Transcription factor Bcl11b sustains iNKT1 and iNKT2 cell programs, restricts iNKT17 cell program, and governs iNKT cell survival

Invariant natural killer T (iNKT) cells are innate-like T cells that recognize glycolipid antigens and play critical roles in regulation of immune responses. Based on expression of the transcription factors (TFs) Tbet, Plzf, and Rorγt, iNKT cells have been classified in effector subsets that emerge in the thymus, namely, iNKT1, iNKT2, and iNKT17. Deficiency in the TF Bcl11b in double-positive (DP) thymocytes has been shown to cause absence of iNKT cells in the thymus and periphery due to defective self glycolipid processing and presentation by DP thymocytes and undefined intrinsic alterations in iNKT precursors. We used a model of cre-mediated postselection deletion of Bcl11b in iNKT cells to determine its intrinsic role in these cells. We found that Bcl11b is expressed equivalently in all three effector iNKT subsets, and its removal caused a reduction in the numbers of iNKT1 and iNKT2 cells, but not in the numbers of iNKT17 cells. Additionally, we show that Bcl11b sustains subset-specific cytokine production by iNKT1 and iNKT2 cells and restricts expression of iNKT17 genes in iNKT1 and iNKT2 subsets, overall restraining the iNKT17 program in iNKT cells. The total numbers of iNKT cells were reduced in the absence of Bcl11b both in the thymus and periphery, associated with the decrease in iNKT1 and iNKT2 cell numbers and decrease in survival, related to changes in survival/apoptosis genes. Thus, these results extend our understanding of the role of Bcl11b in iNKT cells beyond their selection and demonstrate that Bcl11b is a key regulator of iNKT effector subsets, their function, identity, and survival.

NKAP Regulates Invariant NKT Cell Proliferation and Differentiation into ROR-γt-Expressing NKT17 Cells.

Invariant NKT (iNKT) cells are a unique lineage with characteristics of both adaptive and innate lymphocytes, and they recognize glycolipids presented by an MHC class I-like CD1d molecule. During thymic development, iNKT cells also differentiate into NKT1, NKT2, and NKT17 functional subsets that preferentially produce cytokines IFN-γ, IL-4, and IL-17, respectively, upon activation. Newly selected iNKT cells undergo a burst of proliferation, which is defective in mice with a specific deletion of NKAP in the iNKT cell lineage, leading to severe reductions in thymic and peripheral iNKT cell numbers. The decreased cell number is not due to defective homeostasis or increased apoptosis, and it is not rescued by Bcl-xL overexpression. NKAP is also required for differentiation into NKT17 cells, but NKT1 and NKT2 cell development and function are unaffected. This failure in NKT17 development is rescued by transgenic expression of promyelocytic leukemia zinc finger; however, the promyelocytic leukemia zinc finger transgene does not restore iNKT cell numbers or the block in positive selection into the iNKT cell lineage in CD4-cre NKAP conditional knockout mice. Therefore, NKAP regulates multiple steps in iNKT cell development and differentiation.

Syndecan-1 controls the role of iNKT cells in regulating body fat mass and glucose homeostasis in mice

Abstract Obesity is a major risk factor for metabolic disease, with white adipose tissue inflammation emerging as a key underlying pathology that coupled with insulin resistance, type 2 diabetes and cardiovascular diseases. Immune cells play a central role in maintaining immune homeostasis of these adipose tissues. Formerly, significant efforts have been made in understanding the roles of conventional and regulatory T cells. Natural killer T17 cells (NKT17 cells) are lipid-sensing innate T cells, restricted by the antigen-presenting molecule CD1d and express the transcription factor RORyt. Though, the role of invariant natural killer T (iNKT) cells is still remains unclear. Recently, we have discovered that syndecan-1 (sdc1) highly express on the surface of NKT17 cells. After thymus, sdc1-expressing NKT17 cells specifically localize the adipose tissues. Encouraged with previous results, iNKT cells were characterized in both wild type (wt) and sdc1 knock out (sdc1ko) BALB/c mice. The iNKT cells with sdc-1ko mice have shown higher level of proliferation compared to wt. The proliferation assay has shown significantly high number of iNKT cells in S-phase of cell cycle dominating with double positive (CD4+CD8+T cells) T cells in sdc1ko mice. Further, sdc-1ko mice have significantly reduced body fat and improved glucose tolerance. Future study with siRNA, targeting sdc1 in iNKT cells could be a contributing factor in regulating obesity and glucose homeostasis and to understand the physiological significance of sdc1 expression by NKT17 cells. Thus, developing the strategies that maintain immune homeostasis in obese adipose tissue could lead to novel therapeutics that improve and/or prevent the metabolic and cardiovascular risks of obesity.

The Hepatic Recruitment of Innate Natural Killer T cells and Immune-Microenvironment Modulation after Colorectal Cancer Liver Metastasis

Abstract Aims To investigate the modulation of the immune microenvironment by human NKT cells and the crosstalk between NKT cells and Kupffer cells (KC), dendritic cells (DC) in colorectal cancer liver metastasis. Methods The numbers of iNKT cells and other cells from tumor margin, tumor or adjacent normal tissues were analysed by flow cytometry and AMNIS system. Ex vivo co-culture, 3D culture, Elisa array were established for this study. Results In primary colorectal cancers, percentage of iNKT cells was higher in early stage patients (T0~2). In the metastatic CRC, CXCR6+ cells are recruited and enriched at the invasive margin, elevated CXCR6 density was negatively related with Rees’ score, which suggests better prognosis. iNKT cells were the major population (30~40%) of CXCR6+cells, and the proportion and counts of iNKT cells were higher at the invasive margin (<2cm from liver- tumor interphase). CXCL16+ cells were found to be enriched at the invasive margin using immunofluorescence. 85±14% of CXCL16+ (the ligand of CXCR6) cells were CD68+ macrophage, of which 89% cells were Clec4f+Kupffer cells. And the Ex vivo chemotaxis experiments showed that rhCXCL16 and Clec4f+Kupffer cell conditional medium promote iNKT cells recruitment. The ex vivo and in vitro experiments showed that iNKT cells could lead KC to M2 differentiation, however, antigen presented by KC restricted the CRC elimination capability of iNKT cells. In contrast, antigen presenting by DC enhanced clearance of CRC but at meanwhile also induce hepatocytes cell death. Furthermore, we found by in situ staining and FACS that the majority of the KC was M1 KC at intra-tumor area and M2 KC in the adjacent normal tissues. The iNKT cells as well as KC showed Th2 polarization at the invasive margin.

199. Genetic Engineering of Hematopoietic Stem Cells to Generate Invariant Natural Killer T Cells

Invariant natural killer T (iNKT) cells comprise a small population of αβ T lymphocytes. They bridge the innate and adaptive immune systems and mediate strong and rapid responses to many diseases, including cancer, infections, allergies, and autoimmunity. However, the study of iNKT cell biology and the therapeutic applications of these cells are greatly limited by their small numbers in vivo (0.01-1% in mouse and human blood). Here, we report a new method to generate large numbers of iNKT cells in mice through T-cell receptor (TCR) gene engineering of hematopoietic stem cells (HSCs). We showed that iNKT TCR-engineered HSCs could generate a clonal population of iNKT cells. These HSC-engineered iNKT cells displayed the typical iNKT cell phenotype and functionality. They followed a two-stage developmental path, first in thymus and then in the periphery, resembling that of endogenous iNKT cells. When tested in a mouse melanoma lung metastasis model, the HSC-engineered iNKT cells effectively protected mice from tumor metastasis. This method provides a powerful and high-throughput tool to investigate the in vivo development and functionality of clonal iNKT cells in mice. More importantly, this method takes advantage of the self-renewal and longevity of HSCs to generate a long-term supply of engineered iNKT cells, thus opening up a new avenue for iNKT cell-based immunotherapy.

Cellular Adjuvant Properties, Direct Cytotoxicity of Re-differentiated Vα24 Invariant NKT-like Cells from Human Induced Pluripotent Stem Cells.

SummaryVα24 invariant natural killer T (iNKT) cells are a subset of T lymphocytes implicated in the regulation of broad immune responses. They recognize lipid antigens presented by CD1d on antigen-presenting cells and induce both innate and adaptive immune responses, which enhance effective immunity against cancer. Conversely, reduced iNKT cell numbers and function have been observed in many patients with cancer. To recover these numbers, we reprogrammed human iNKT cells to pluripotency and then re-differentiated them into regenerated iNKT cells in vitro through an IL-7/IL-15-based optimized cytokine combination. The re-differentiated iNKT cells showed proliferation and IFN-γ production in response to α-galactosylceramide, induced dendritic cell maturation and downstream activation of both cytotoxic T lymphocytes and NK cells, and exhibited NKG2D- and DNAM-1-mediated NK cell-like cytotoxicity against cancer cell lines. The immunological features of re-differentiated iNKT cells and their unlimited availability from induced pluripotent stem cells offer a potentially effective immunotherapy against cancer.

CDR3β sequence motifs regulate autoreactivity of human invariant NKT cell receptors

Invariant natural killer T (iNKT) cells are a subset of T lymphocytes that recognize lipid ligands presented by monomorphic CD1d. Human iNKT T cell receptor (TCR) is largely composed of invariant Vα24 (Vα24i) TCRα chain and semi-variant Vβ11 TCRβ chain, where complementarity-determining region (CDR)3β is the sole variable region. One of the characteristic features of iNKT cells is that they retain autoreactivity even after the thymic selection. However, the molecular features of human iNKT TCR CDR3β sequences that regulate autoreactivity remain unknown. Since the numbers of iNKT cells with detectable autoreactivity in peripheral blood is limited, we introduced the Vα24i gene into peripheral T cells and generated a de novo human iNKT TCR repertoire. By stimulating the transfected T cells with artificial antigen presenting cells (aAPCs) presenting self-ligands, we enriched strongly autoreactive iNKT TCRs and isolated a large panel of human iNKT TCRs with a broad range autoreactivity. From this panel of unique iNKT TCRs, we deciphered three CDR3β sequence motifs frequently encoded by strongly-autoreactive iNKT TCRs: a VD region with 2 or more acidic amino acids, usage of the Jβ2-5 allele, and a CDR3β region of 13 amino acids in length. iNKT TCRs encoding 2 or 3 sequence motifs also exhibit higher autoreactivity than those encoding 0 or 1 motifs. These data facilitate our understanding of the molecular basis for human iNKT cell autoreactivity involved in immune responses associated with human disease.

Larger Number of Invariant Natural Killer T-Cells in Allogeneic Peripheral Blood Stem Cell Grafts Is Associated with Improved Graft-Versus-Host Disease-Free, Progression-Free Survival after Allogeneic Stem Cell Transplantation

IntroductionThe therapeutic efficacy of allogeneic stem cell transplantation (allo-SCT) relies mostly on the immune-mediated graft-versus-tumor (GVT) activity of the graft. However the beneficial effect of GVT is counterbalanced by the destruction of the tissues of the recipient by the immune effectors of the donor, termed graft-versus-host disease (GVHD). Despite G-CSF mobilized peripheral blood stem cells (PBSC) being used for more than 20 years, literature investigating the impact of the different graft cell subsets on outcome, remains scarce. Therefore, this study aimed to investigate the impact of a comprehensive set of immune cells contained within PBSC grafts on patients' outcome after allo-SCT, using the composite endpoint of GVHD-free and progression-free survival (GPFS) as primary endpoint. Indeed, some cell subset may lead to a decreased incidence of relapse but at the expense of an increased incidence of GVHD or vice versa. The use of the GPFS composite endpoint would allow to overcome this limitation, identifying cellular subsets that correlate with cure without ongoing morbidity.Patients and methods.80 consecutive patients who underwent allo-SCT in a single center using G-CSF-mobilized PBSCs between 2010 and 2013 and for whom frozen aliquots of the PBSC grafts were available, were included in this study. Immune cell subset quantification was performed by multi-color flow cytometry. Patients median age was 57 years, 57% of patients had a myeloid malignancy and 43% a lymphoid malignancy. Disease risk index was low or intermediate in 79% of patients and high or very high in 21% of patients. The majority of patients received a reduced-intensity conditioning regimen (81%) and in-vivo T-cell depletion using anti-thymocyte globulin (ATG) (91%). GVHD prophylaxis was cyclosporine alone (34%), or in combination with either mycophenolate mofetil (59%) or methotrexate (7%). 65% of patients received grafts from unrelated donors and 35% from matched related donors.Naïve and memory T cell and B cell subsets, regulatory T cells, invariant natural killer T-cells (iNKT), natural killer cells (NK) and dendritic cell subsets were analyzed. Cell subsets were selected for the purpose of this study based on the absence of a strong correlation with another graft subset (Pearson or Spearman correlation >0.8). P-values were not adjusted for multiple comparisons, but only covariates for which the q value is <0.2 are presented. GPFS was defined as survival without disease progression, grade III-IV acute GVHD or chronic GVHD requiring systemic therapy.ResultsMedian follow-up was 36 months. The median numbers of nucleated cells, CD34+ cells, and total T-cells in the allograft were 102.9, 7.2, and 20.7 x 106/kg, respectively. The numbers of nucleated cells, CD34+ cells, T-cells, B-cells, NK cells, and dendritic cells in the PBSC graft, were not significantly associated with overall survival (OS) and GPFS. Invariant NKT cells were the only cell subset associated with GPFS with univariate p-value <0.01 and q values <0.2. Stratifying the entire group of transplant recipients by median value for iNKT cells (median number 0.011x106/kg) showed better GPFS among patients receiving larger number of iNKT cells, being 49% at 2 years, versus 22% for patients receiving <0.011x106/kg iNKT/Kg (p=0.007) (Figure 1). In multivariate analysis, adjusted for patient age, type of donor, disease risk index, GVHD prophylaxis, conditioning regimen intensity and use of ATG, dose of iNKT cells was the only parameter with a significant impact on GPFS (hazard ratio: 0.45, 95% CI: 0.25-0.80, p=0.007). Transplantation with more than the median number of iNKT cells was also associated with a trend toward an improved progression-free survival (59% versus 39% at 2 years, p=0.12) and a decreased risk of extensive chronic GVHD (5.1% versus 17.5% at 2 years, p=0.07). In contrast, the number of iNKT cells transplanted has no impact on grade II-IV aGVHD (10% versus 15%, p=0.49) or overall survival (60% versus 55%, p=0.51).ConclusionAmong patients undergoing allo-SCT and receiving a PBSC graft, a higher number of iNKT, a regulatory cell population implicated in immune tolerance and tumor surveillance, is associated with an improved GPFS. These data may pave the way for prospective and active interventions aiming to manipulate the graft content in order to improve allo-SCT outcome. [Display omitted] DisclosuresMoreau:Millennium: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen-Cilag: Honoraria, Membership on an entity's Board of Directors or advisory committees. Mohty:Janssen: Honoraria; Celgene: Honoraria.

NKTT120 Safely Depletes iNKT Cells in Stable Adult Sickle Cell Patients in a Phase 1 Trial

Patients with sickle cell disease have an increased number of circulating activated iNKT cells while murine SCD models report increased number and activation state of iNKT cells in target organs. Furthermore, the use of a murine iNKT cell-depleting antibody in murine SCD models prevents inflammation driven end-organ damage. NKTT120 is a humanized monoclonal antibody directed to the unique invariant TCR of iNKT cells that depletes these cells by ADCC. In preclinical studies, NKTT120 has demonstrated a safe and specific dose and time dependent depletion/recovery of iNKT cells. The preclinical efficacy and safety data supported a clinical development program to show that NKTT120 demonstrates the same safety and specificity for iNKT cell depletion from the peripheral circulation in SCD patients. In this first in human phase 1 dose-escalation study, we have examined the safety of NKTT120 in adults with steady state SCD. Future studies will explore the ability of NKTT120 prevent painful vaso-occlusive crises.Objective: To determine the safety, maximum tolerated dose (MTD), pharmacokinetics, and pharmacodynamics of NKTT120 in adults with steady state SCD. The optimal dose for a phase 2 study of NKTT120 will deplete iNKT for approximately 3 months allowing for periodic dosing.Methods: A first-in-humanphase 1 study utilizing a 3+3 design to evaluate single doses escalating over a range of 7 doses from 0.001 mg/kg to 1.0 mg/kg. The primary outcome measure is safety. Secondary outcomes include blood iNKT cell depletion and recovery, pain, analgesic use, quality of life (QoL), and pulmonary function. During a screening run-in period and after dosing of NKTT120, subjects maintained a daily smartphone eDiary (eSCaPe) to report pain, respiratory symptoms and analgesic use. ASCQ-Me and PROMIS QoL questionnaires were administered at clinic visits. The screening run-in outcomes will be used as baseline comparison for values obtained post-dosing.Results: A total of 21 patients were enrolled into the 7 cohorts of the completed and closed study. The drug was delivered as a 10-minute IV push in all cohorts. No MTD was defined, as no DLTs were reported. Three subjects each were dosed at 0.001, 0.003, 0.01, 0.03, 0.1, 0.3 or 1.0 mg/kg. At leastone month of follow-up data on circulating iNKT cell numbers are available for all of the patients dosed in the study. Only iNKT cell counts were affected by NKTT120 dosing, no change in other hematologic parameters was observed in peripheral blood. No acute elevation in circulating inflammatory cytokines was seen after antibody administration. All doses of NKTT120 resulted in maximum depletion of iNKT cells at the first time point (6 hours) monitored in all patients. During the recovery period, all patients had detectable iNKT cells in their peripheral blood. In all cohorts, the time to recovery of iNKT cells correlates with the starting circulating levels, with a longer recovery time for patients with lower baseline cell numbers. T1/2 is approximately 11 days. As observed in the pre-clinical safety studies, iNKT cell depletion and recovery was dose and time dependent. At the recommended Phase 2 dose (0.3 mg/kg) no iNKT cells were detectable in the peripheral circulation for a period of several months, suggesting near complete tissue depletion at these doses requiring recovery from T cell precursors that are not targeted by NKTT120.Conclusions: In adults with SCD,NKTT120 administered up to a dose of 1.0 mg/kg specifically reduces iNKT cells without NKTT120 dose limiting toxicity. Patients at the higher dose cohorts of NKTT120 illustrate temporal pattern for iNKT cell depletion and recovery in the circulation that inform the dosing strategy for phase 2 studies. The recommended Phase 2 dose is 0.3 mg/kg administered at a 3 month interval. The Phase 2 study will highlight the reduction of iNKT cells in the suppression of the inflammatory stimuli that promote many of the pathophysiologic sequelae seen in SCD. DisclosuresEaton:NKT Therapeutics: Employment. Mazanet:NKT Therapeutics: Consultancy, Equity Ownership. Nathan:NKT Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Pharmacokinetics and Pharmacodynamics after Subcutaneous Dosing of the iNKT Cell Depleting Antibody NKTT120

The iNKT cell (invariant Natural Killer T cell) is a unique immune cell that has been shown to be associated with chronic inflammation in sickle cell disease. One approach to reducing iNKT mediated inflammation would be to reduce the number of iNKT cells in the tissue of SCD patients. NKTT120 is a humanized monoclonal antibody directed to the unique T cell receptor of invariant NKT cells that can deplete iNKT cells by antibody dependent cellular cytotoxicity. It recently completed a first in man Phase 1 study that demonstrated that intravenous single doses in adults with SCD specifically reduced iNKT cells without dose limiting toxicity in the therapeutic range of 0.3 and 1.0 mg/kg.Although intravenous dosing was found to be effective and safe, the ability to dose patients by subcutaneous administration would be an asset to a chronic treatment strategy and patient compliance. The purpose of this study was to assess the bioavailability and the efficacy of NKTT120 administered subcutaneously (SC) to cynomolgus monkeys. Female adult cynomolgus monkeys were administered NKTT120 as a single subcutaneous dose of 0.03, 0.1, 0.3 mg/kg (n=3 each) or as a single intravenous (IV) dose of 0.3 mg/kg (n=3). Animals were monitored for iNKT cell depletion of peripheral blood by FACS analysis and NKTT120 concentration was monitored by ELISA.All doses resulted in complete depletion of blood iNKT cells without any change in other blood cells. The return of measureable peripheral blood iNKT cells occurred in each group when the plasma concentration was ≤ 100 ng/mL. The time to recovery was dose dependent with measureable iNKT cells found at 4-6 weeks with the 0.03 mg/kg dose and 8-14 weeks with the 0.3 mg/kg IV and SC doses. NKTT120 was dose proportional with time to maximum plasma concentration of 48 hours following SC dosing. Bioavailability of NKTT120 after SC dosing was ~60% and T1/2 was 15 days for the IV dose group and ranged from 11-19 days for the SC dose groups. The duration of iNKT cell depletion and time to recovery for the 0.3 mg/kg SC dose was similar to that observed for the 0.3 mg/kg IV dose group.The results support a SC dosing strategy for NKTT120 and suggest that similar pharmacodynamic responses could be expected when a therapeutic intravenous dose (0.3 mg/kg) is administered by the subcutaneous route in SCD patients every 3-4 months.Plasma Concentration vs. Time for NKTT120 Following IV or SC doses to Cynomolgus Monkeys [Display omitted] [Display omitted] DisclosuresSchaub:NKT Therapeutics: Employment, Equity Ownership. Thariath:NKT Therapeutics: Employment, Equity Ownership. Scheuplein:NKT Therapeutics: Equity Ownership. Mashal:NKT Therapeutics: Employment, Equity Ownership.

Increased Intraepithelial Vα24 Invariant NKT Cells in the Celiac Duodenum.

Celiac Disease (CD) is an interferon (IFN)γ-mediated duodenal hypersensitivity to wheat gluten occurring in genetically predisposed individuals. Gluten-free diet (GFD) leads to a complete remission of the disease. Vα24-restricted invariant NKT (iNKT) cells are important to maintain immune homeostasis in the gut mucosa because of their unique capacity to rapidly produce large quantities of both T-helper (Th)1 and Th2 cytokines upon stimulation. We studied the presence of these cells in the CD duodenum. Duodenal biopsies were obtained from 45 untreated-CD patients (uCD), 15 Gluten Free Diet-CD patients (GFD-CD), 44 non-inflamed non-CD controls (C-controls) and 15 inflamed non-CD controls (I-controls). Two populations from Spain and Argentina were recruited. Messenger RNA (mRNA) expression of Vα24-Jα18 (invariant TCRα chain of human iNKT cells), IFNγ and intracellular transcription factor Forkhead Box P3 (Foxp3), and flow cytometry intraepithelial lymphocyte (IEL) profile were determined. Both uCD and GFD-CD patients had higher Vα24-Jα18 mRNA levels than non-CD controls (I and C-controls). The expression of Vα24-Jα18 correlated with Marsh score for the severity of mucosal lesion and also with increased mRNA IFNγ levels. uCD and GFD-CD patients had decreased mRNA expression of FoxP3 but increased expression of Vα24-Jα18, which revealed a CD-like molecular profile. Increased numbers of iNKT cells were confirmed by flow cytometry within the intraepithelial lymphocyte compartment of uCD and GFD-CD patients and correlated with Vα24-Jα18 mRNA expression. In conclusion, we have found an increased number of iNKT cells in the duodenum from both uCD and GFD-CD patients, irrespective of the mucosal status. A CD-like molecular profile, defined by an increased mRNA expression of Vα24-Jα18 together with a decreased expression of FoxP3, may represent a pro-inflammatory signature of the CD duodenum.