TLR3 Mediates Senescence and Immunosurveillance of Hepatic Stellate Cells

Background: Activation of hepatic stellate cells (HSCs) is an important driver of liver fibrosis, which is a health problem of global concern, and there is no effective solution for it at the present. Senescent activated HSCs are preferentially killed by natural killer cells (NK cells) to promote the regression of hepatic fibrosis. Objectives: The purpose of this study was to investigate the effect of polyinosinic-polycytidylic acid (poly I:C) on HSCs’ senescence, a trigger for NK cell-induced cytotoxicity. Methods: The senescence of HSCs was assessed by western blot, qRT-PCR, and flow cytometry, and NK cell cytotoxicity was assessed in a co-culture of NK cells with poly I:C-treated HSCs by measuring CD107a expression. Results: The expression of p16, p21, SA-β-gal, MICA/MICB, and ULBP2 increased in poly I:C-treated HSCs, rendering them significantly susceptible to NK cell cytotoxicity. Conclusions: Poly I:C induces cellular senescence in HSCs and triggers NK cell immunosurveillance, suggesting that the role of poly I:C in HSC senescence may promote fibrosis regression.

Evaluation of the Expression of CCR5 and CX3CR1 Receptors and Correlation with the Functionality of T Cells in Women infected with ZIKV during Pregnancy

There have been reports of neurological abnormalities associated with the Zika virus (ZIKV), such as congenital Zika syndrome (CZS) in children born to mothers infected during pregnancy. We investigated how the immune response to ZIKV during pregnancy is primed and conduct a thorough evaluation of the inflammatory and cytotoxic profiles as well as the expression of CCR5 and CX3CR1. We compared the reactivity of T cells to ZIKV peptides in convalescent mothers infected during pregnancy. The child’s clinical outcome (i.e., born with or without CZS) was taken to be the variable. The cells were stimulated in vitro with ZIKV peptides and evaluated using the ELISPOT and flow cytometry assays. After in vitro stimulation with ZIKV peptides, we observed a tendency toward a higher Interferon gamma (IFN-γ)-producing T cell responses in mothers who had asymptomatic children and a higher CD107a expression in T cells in mothers who had children with CZS. We found a higher frequency of T cells expressing CD107a+ and co-expressing CX3CR1+CCR5+, which is much clearer in the T cells of mothers who had CZS children. We suggest that this differential profile influenced the clinical outcome of babies. These data need to be further investigated, including the evaluation of other ZIKV peptides and markers and functional assays.

Human mucosal-associated invariant T cells respond to Mucorales species in a MR1-dependent manner

Activation of mucosal-associated invariant T cells (MAIT cells) by certain bacteria, viruses, and yeast is well studied, but the activation potential of filamentous moulds from the order Mucorales is not known. Here, we show a rapid response of human MAIT cells against the Mucorales species Mucor circinelloides, Rhizopus arrhizus, and Rhizopus microsporus. This activation included upregulation of CD69 and degranulation marked by increased CD107a expression, while intracellular perforin and granzyme A expression were reduced. Furthermore, blocking of the antigen-presenting molecule major histocompatibility complex class I-related abrogated MAIT cell activation demonstrating a T cell receptor-dependent stimulation by Mucorales.

Obinutuzumab-Retreatment in Combination with Bendamustine Is Effective for Obinutuzumab Resistant Models in Vitro and In Vivo

Background: Follicular lymphoma (FL) commonly recurs and is difficult to cure. Obinutuzumab (OBI) is a humanized type II anti-CD20 antibody. It's mode of action is mainly characterized by the induction of direct cell death and it shows stronger antibody-dependent cellular cytotoxicity (ADCC) than rituximab. While OBI is indicated for previously untreated or relapsed/refractory (r/r) FL, there is no evidence on the efficacy of retreatment with OBI in r/r FL after prior OBI-containing therapy. To demonstrate the effectiveness of OBI-retreatment in a non-clinical study, we established in vitro and in vivo OBI resistant models, consisting of human non-Hodgkin lymphoma (NHL) cells resistant to OBI-induced ADCC and OBI-resistant tumors established from NHL cells under xenotransplantation conditions, and investigated the combination efficacy of OBI and bendamustine (Benda) in these OBI-resistant models. Methods: OBI-induced ADCC resistant clones were established from an RL cell line by inducing ADCC three times with CD16-transfected NK-92 cells. The in vivo resistant model was established from a SU-DHL-4 xenograft model through repeated treatment with OBI and re-inoculation of the regrown tumors. CD20 expression was assessed by flow cytometry or immunohistochemistry (IHC). ADCC activity was evaluated using calcein-AM with CD16-transfected NK-92 cells. To examine antitumor activity, OBI, human immunoglobulin G (HuIgG), Benda, or vehicle were intravenously administered on Day 1, 8 and 15 (OBI and HuIgG) or Day 1 and 2 (Benda and vehicle), and tumor volume was measured. Intratumorally infiltrated NK cells were assessed by IHC of CD335. Cell surface CD107a expression was assessed by flow cytometry. Results: OBI-induced ADCC resistant clones derived from RL (RL-OR-8 and RL-OR-22) showed a reduction in ADCC sensitivity compared with RL. These resistant clones exhibited CD20 expression similar to RL. Pretreatment of the effector NK cells with Benda enhanced ADCC induction of OBI in RL-OR-8 and RL-OR-22. Treatment with OBI (30 mg/kg) in combination with Benda (13.3 mg/kg) also significantly increased antitumor activity compared with each single agent alone on Day 18 (when tumor volume reached euthanasia criteria (*) in control and OBI monotherapy groups) in the RL-OR-22 xenograft model (Table 1). An in vivo OBI resistant model derived from SU-DHL-4 xenografts (SU-DHL-4-OR-18-8) was also established. While SU-DHL-4 xenografted tumors disappeared in 6/6 mice after the third OBI treatment (6 mg/kg), SU-DHL-4-OR-18-8 tumors did not regress in 6/6 mice. CD20 expression in SU-DHL-4-OR-18-8 tumors did not decrease compared with SU-DHL-4 tumors. The ratio of CD335-positive cells to tumor cells after 6 mg/kg of OBI treatment (Day 4) in SU-DHL-4-OR-18-8 was significantly decreased compared with the ratio in SU-DHL-4 (0.31 ± 0.12% vs 0.93 ± 0.23%). The combination efficacy of OBI and Benda was also assessed in an SU-DHL-4-OR-18-8 xenograft model. OBI (6 mg/kg) in combination with Benda (25 mg/kg) significantly increased the antitumor activity compared with each single agent alone on Day 22 (*in control group) and 29 (Table 1). Finally, the expression of CD107a, an NK cell-degranulation marker, was detected to examine the effect of Benda on NK activity in vitro. Pretreating effector NK cells with Benda upregulated CD107a expression on NK cell surfaces after OBI treatment in RL-OR-8 and RL-OR-22. Conclusions: The decreased CD335-positive cell ratio observed in the in vivo SU-DHL-4-OR-18-8 resistant model suggests that the mechanism of resistance to OBI also involves the attenuation of ADCC as in the in vitro OBI-induced ADCC resistant clones. The combination treatment of OBI and Benda was effective in both RL-OR-22 and SU-DHL-4-OR-18-8 xenograft models. It is possible that activation of NK cells by Benda might be involved in this combination mechanism. Although the mechanisms need to be examined in more detail, these results indicate the possible effectiveness of OBI-retreatment after prior OBI-containing therapy. Disclosures Yamashita-Kashima: Nippon Shinyaku Co., Ltd.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Current Employment. Kawasaki:Chugai Pharmaceutical Co., Ltd.: Current Employment; Nippon Shinyaku Co., Ltd.: Research Funding. Yorozu:Chugai Pharmaceutical Co., Ltd.: Current Employment; Nippon Shinyaku Co., Ltd.: Research Funding. Yoshiura:Chugai Pharmaceutical Co., Ltd.: Current Employment; Nippon Shinyaku Co., Ltd.: Research Funding. Fujimura:Chugai Pharmaceutical Co., Ltd.: Current Employment; Nippon Shinyaku Co., Ltd.: Research Funding. Kurasawa:Chugai Pharmaceutical Co., Ltd.: Current Employment; Nippon Shinyaku Co., Ltd.: Research Funding. Harada:Chugai Pharmaceutical Co., Ltd.: Current Employment; Nippon Shinyaku Co., Ltd.: Research Funding. Yoshimura:Nippon Shinyaku Co., Ltd.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Current Employment.

IMMU-47. PROTEIN PHOSPHATASE 2A INHIBITION IN NK CELL THERAPY FOR TREATMENT OF MEDULLOBLASTOMA

Checkpoint immunotherapy (ICB) thus far has shown limited efficacy against brain tumors, such as medulloblastoma (MB). Its low mutational burden is thought to result in a paucity of neoantigen to trigger an effective T-cell response. Natural killer (NK) cells, can recognize tumor cells independently of neoantigens, making them appealing against MBs. Modulation of NK cells to enhance cytotoxicity against MBs could be a novel treatment strategy. Protein Phosphatase 2A (PP2A), a ubiquitous serine/threonine phosphatase, has been shown to inhibit IFNg and Granzyme B production by NK cells. We hypothesize that NK92, a transformed human NK cell line, has intrinsic activity against human MB cells and that inhibiting PP2A pharmacologically can enhance cytotoxicity of NK92 cells. We performed NK cytotoxicity assay and granulation assay against human MB cell line D425. We also used a small molecular inhibitor, LB100, to modulate PP2A activity in NK92. NK92 cells were co-cultured with D425, in increasing E:T (Effector:Target) ratio for 4 hours. D425 cells were pre-labeled with CellTrace Violet dye. The percentage of D425 (Violet+) cells in apoptosis (Cas3/7+) or necrosis (AAD+) were compared with different ET ratios to quantify NK mediated cell cytotoxicity. We also measured CD107a expression in NK92 to assess granulation with LB100 treatment. D425 cells were sensitive to NK92 killing. Percentage of D425 cells either apoptotic or necrotic increased with increasing ET ratio, suggesting that there was NK92 mediated cytotoxicity. Percentage of killed D425 cells ranged from 18% at baseline (without NK92) to 80% at ET ratio of 20. Inhibition of PP2A using LB100, enhanced NK92 degranulation. CD107a+ NK92 cells increased from 19% to 28% with 8uM of LB100. NK92 cells are cytotoxic against MB cells in vitro and inhibition of PP2A in NK cells can enhance their activity against MB cells.

The potential target of double negative T cells in cancer immunotherapy.

e15180 Background: Double negative T cell (DNT) is defined by the expression of CD3 (or TCRαβ) with the absence of CD4, CD8 and iNKT cell markers. DNTs account for 1-3% of the total peripheral lymphocytes. Although healthy donor cells derived allogeneic DNTs were reported to have anti-tumor effects in several cancer models, the function of DNTs in tumor microenvironment is not well understood. In this study, we aim to characterize DNTs in cancer tissues and lymph nodes of colorectal cancer patients including their immunological functions. Methods: A total of 14 colorectal cancer tissues and regional lymph nodes were collected and examined for the proportion and the immunological functions of DNTs by FACS analysis. Results: DNTs, which were defined in this study as TCRαβ positive with CD4-, CD8- and CD56- T cells, were detected in colorectal cancer tissues. DNT cells account for more than 5% of TCRαβ-positive populations in 12 cases and out of which 7 cases were more than 20% observed in this study. In all of these 7 cases, DNT cells were also found to be higher in the tumor tissues than in non-metastatic lymph nodes. In addition to colorectal cancer, DNTs were found to be more than 5% of TCRαβ-positive populations in cancer tissues in 3 out of the 6 cases of ovarian and uterine cancer cases.To further evaluate the potential immunological functions of DNTs, expression of cell membrane protein markers were evaluated. CD3ε expression was down regulated in almost all of the DNTs. The CD107a expression of DNTs were higher than that of CD8+ T cells in tumor tissues. When co-culturing DNTs with tumor cells, CD107a expression of DNTs was significantly upregulated. Notably, the intracellular production of perforin and granzyme B were lower than CD8+ T cells. Fas-L expression were found to be higher on DNT cells compared to other T cell populations, suggesting DNT might possess cytotoxic activity in the tumor microenvironment. It is known that upregulation of Fas expression is caused by T cell activation and subsequently induces T cell apoptosis. The upregulation of Fas expression was also observed in DNT cells from non-metastatic lymph nodes. In contrast, Fas expression of DNT cells in tumor tissues was observed to be lower than CD4+ or CD8+ T cells, suggesting that DNT could escape from apoptosis. This downregulation of Fas expression was speculated to be one of the reasons for the accumulation of DNTs in tumor tissues. Conclusions: Although the function of DNTs was still not well elucidated, the accumulation of DNT cells in tumor microenvironment suggests DNTs could be a potential target for cancer immunotherapy.

NKG2C+CD57+ Natural Killer Cells May Play an Important Role in Maintaining Virus-Specific Immune Reconstitution Post-Allogeneic HSCT

Background: Allogeneic hematopoietic stem cells transplantation (HSCT) is the only possible curative treatment for several malignant hematological diseases in adults. The delayed immune reconstitution after HSCT can allow human cytomegalovirus (CMV) to reactivate. This leads to prolonged hospitalization, increased morbidity and even mortality. Natural Killer (NK) cells have been described to undergo a persistent reconfiguration in response to CMV-reactivation. Here we analyzed the presence and expansion of adaptive-NK cells in patients after allogeneic HSCT. Methods: A multicolor flow cytometry panel for monitoring the adaptive-NK cell (NKG2C+CD57+) reconstitution was established. Reconstitution of adaptive-NK cells was assessed in peripheral blood samples from 70 CMV-seropositive patients between day 0 and 100 post-HSCT at intervals of 7-10 days. Monitoring of CMV-reactivation was determined by CMV-pp65 antigenemia and reconstitution of CMV-specific T cells (CMV-CTLs) was performed routinely using 7 commercially available, certified CMV-tetramers. For further immunological tests, PBMCs from 7 patients were isolated by density gradient centrifugation. Total NK cells were negatively selected by magnetic bead separation. Additional purification of adaptive-NK cellswas achieved by cell sorting. Selected adaptive-NK cells were expanded by co-culture with irradiated allogeneic PBMCs as feeder cells and the medium was supplemented with PHA, IL-2 and IL-15. Cytotoxicity assays were performed with expanded adaptive-NK cells co-cultured for 5h with CMV-infected human fibroblasts (HFF) or K562 cell line. Degranulation capacity was assessed by CD107a staining and the cytotoxic effect was assessed by lactate dehydrogenase (LDH) assay. Additionally, T cells capacity to migrate towards adaptive-NK cells co-cultured with or without CMV-infected HFF was evaluated using 5.0 µm polycarbonate membranes. Results: Our patient cohort consisted of 70 patients after allogeneic HSCT with a median age of 58 years (range: 19-73). Forty-six patients (65.7%) were transplanted for acute leukemia, 58 (82.9%) received reduced intensity conditioning (RIC) and 67 (95.7%) received anti-thymocyte-antibodies globulin (ATG). GvHD-prophylaxis was cyclosporine A (CsA) in combination with mycophenolate motefil (MMF) for 77.1% of the patients and 80% were transplanted from matched donors. Thirty-seven (52.9%) patients reactivated CMV (median age: 61 years, range 23-73; median day of reactivation: day +37 post-HSCT, range: 17-63). A significant increase in the absolute cell counts of adaptive-NK cells was observed after CMV reactivation, when compared to patients who did not reactivate CMV (p<0.001). Degranulation capacity of in vitro expanded adaptive-NK cells (up to 3680 fold-expansion) was analysed by CD107a staining after co-culture with CMV-infected HFF at three different Effector : Target ratios (E:T) of 1:1; 1:2; 1:5. A statistically significant increase in CD107a expression in adaptive-NK cells was seen for all E:T ratios. In addition, this CD107a expression was not statistically different from the positive control. Furthermore, LDH colorimetric assay was used to supplement the information on cytotoxic capacity of adaptive-NK cells. A significant increase of LDH release was seen in CMV-infected HFF when compared to uninfected HFF (p<0.0001). When comparing adaptive-NK cells with CMV-CTLs, both cell populations show similar kinetics of expansion during CMV reactivation, with an increase in absolute counts. Surprisingly however, adaptive-NK cells continue to expand even after resolution of CMV-reactivation, while CMV-CTLs cell pool starts to retract (p<0.05). We then evaluated the capacity of adaptive-NK cells to recruit T cells. We observed a significantly increased fold difference in the migration of T cells when adaptive-NK cells were co-culture with CMV-infected HFF in comparison to T cell chemoattractant CCL21 (p<0.05). Conclusion: Taken together, our results indicate that adaptive-NK cells can undergo a dynamic modulation in response to CMV-reactivation. This cell population is not only able to eliminate CMV-infected target cells but can also recruit T cells to the site of infection. It is possible that adaptive-NK cells may substitute for missing CMV-CTLs shortly after HSCT and may also maintain a memory phenotype of CMV-CTLs. Disclosures No relevant conflicts of interest to declare.

Subclinical Cytomegalovirus DNA Is Associated with CD4 T Cell Activation and Impaired CD8 T Cell CD107a Expression in People Living with HIV despite Early Antiretroviral Therapy

ABSTRACTMost people living with HIV (PLWH) are coinfected with cytomegalovirus (CMV). Subclinical CMV replication is associated with immune dysfunction and with increased HIV DNA in antiretroviral therapy (ART)-naive and -suppressed PLWH. To identify immunological mechanisms by which CMV could favor HIV persistence, we analyzed 181 peripheral blood mononuclear cell (PBMC) samples from 64 PLWH starting ART during early HIV infection with subsequent virologic suppression up to 58 months. In each sample, we measured levels of CMV and Epstein-Barr virus (EBV) DNA by droplet digital PCR (ddPCR). We also measured expression of immunological markers for activation (HLA-DR+CD38+), cycling (Ki-67+), degranulation (CD107a+), and the immune checkpoint protein PD-1 on CD4+and CD8+T cell memory subsets. Significant differences in percentages of lymphocyte markers by CMV/EBV shedding were identified using generalized linear mixed-effects models. Overall, CMV DNA was detected at 60/181 time points. At the time of ART initiation, the presence of detectable CMV DNA was associated with increased CD4+T cell activation and CD107a expression and with increased CD8+T cellular cycling and reduced CD107a expression on CD8+T cells. While some effects disappeared during ART, greater CD4+T cell activation and reduced CD107a expression on CD8+T cells persisted when CMV was present (P < 0.01). In contrast, EBV was not associated with any immunological differences. Among the covariates, peak HIV RNA and CD4/CD8 ratio had the most significant effect on the immune system. In conclusion, our study identified immune differences in PLWH with detectable CMV starting early ART, which may represent an additional hurdle for HIV cure efforts.IMPORTANCEChronic viral infections such as with HIV and CMV last a lifetime and can continually antagonize the immune system. Both viruses are associated with higher expression of inflammation markers, and recent evidence suggests that CMV may complicate efforts to deplete HIV reservoirs. Our group and others have shown that CMV shedding is associated with a larger HIV reservoir. Subclinical CMV replication could favor HIV persistence via bystander effects on our immune system. In this study, we collected longitudinal PBMC samples from people starting ART and measured immune changes associated with detectable CMV. We found that when CMV was detectable, CD4+T cell activation was higher and CD8+T cell degranulation was lower. Both results may contribute to the slower decay of the size of the reservoir during CMV replication, since activated CD4+T cells are more vulnerable to HIV infection, while the loss of CD8+T cell degranulation may impede the proper killing of infected cells.

Cyclin-Dependent Kinases (CDK) Signaling Blockade Potentiates NK Cell Mediated Cytotoxicity Against Acute Myelogenous Leukemia

Dinaciclib, a CDK 1, 2, 5, and 9 inhibitor, has been shown to have potent anti-cancer activity against several malignancies. Our group previously demonstrated a direct RALB-dependent proapoptotic activity of dinaciclib against acute myeloid leukemia (AML) (Oncogene (2017) 36, 3263-3273). Furthermore, there is emerging evidence that dinaciclib can influence the recognition and killing of cancer cells by the immune system suggesting that it could have multimodal anti-cancer activities. As natural killer (NK) cells have a critical role in antineoplastic cytotoxicity, we hypothesized that dinaciclib may influence NK cell mediated cytotoxicity against human AML cells. To address this hypothesis, we evaluated the effects of dinaciclib on the NK cytotoxicity against human AML target cells and expression of known regulators of NK cell function. Specifically, we treated THP-1 and KG-1 cells with vehicle or dinaciclib at 5-20 nM for 24 hours, then washed the AML targets and added purified NK cells from PBMCs of healthy donors at an effector: target (E:T) ratio of 2:1. After 24 hours, viable AML target cells were enumerated using Fixable Aqua Dead Cell Stain and AccuCheck Counting Beads (Thermo Fisher Scientific) by flow cytometry. Relative NK cell target killing (%) was calculated by comparing the percentage of AML targets killed with drug treatment combined with donor NK cells relative to drug treatment alone in the absence of NK cells (Relative NK killing %). We assessed NK cell activation by measuring intracellular interferon-ɣ production and CD107a expression as well as NK ligand expression on dinaciclib-treated AML targets using flow cytometry. Dinaciclib treatment of AML targets enhanced the relative NK killing % for both KG-1 and THP-1 AML targets compared to control treatment (for KG-1, 45.6% vs 14.5%, p=0.05; for THP-1, 63.7% vs 31.7%, p=0.01) (Figure 1A, 1B) and was associated with enhanced NK cell degranulation as measured by CD107a expression (DMSO group, 8.3% vs dinaciclib 20nM group, 17.7%, p=0.002) and inflammatory cytokine production as measured by intracellular interferon-ɣ expression (DMSO group, 10.4% vs dinaciclib 20nM group 17.3%, p=0.01) (Figure 1C, 1D) supporting the ability of dinaciclib to sensitize AML targets for NK cell-based immunotherapy. To investigate potential mechanisms that promote NK cell activation by dinaciclib-treated AML targets, we assessed expression of several key NK cell ligands on AML targets and found that dinaciclib treatment led to decreased expression of inhibitory ligands including HLA class I, CD112, CD155 and increased expression in activating ligands such as TRAILR1, CD48 (Figure 1E, 1F) providing potential mechanistic insights. Notably, preliminary studies using a primary, patient-derived AML sample treated with dinaciclib resulted in a similar increase in the proportion of CD107a+ NK cells compared to the control group (44.7% increase, p=0.02, Figure 1G, 1H). While the detailed mechanisms for our findings remain to be determined, this is the first report to our knowledge that inhibiting CDK signaling can sensitize AML targets to NK cell based immunotherapy, a completely novel treatment approach. Further studies are ongoing to investigate the potential for combined targeted therapy with dinaciclib and adoptive NK cell therapy including patient derived xenograft (PDX) mice to validate its translational potential. Disclosures Felices: GT Biopharma: Research Funding.

Perforin and CD107a testing is superior to NK cell function testing for screening patients for genetic HLH

Key Points NK cell function testing is less sensitive and no more specific for discriminating genetic HLH compared to perforin and CD107a expression. Perforin and CD107a testing could augment NK-cell cytotoxicity testing for use in HLH diagnostic criteria.