Loss of CCR7 Expression on CD56bright NK Cells Is Associated with a CD56dimCD16+ NK Cell-Like Phenotype and Correlates with HIV Viral Load

NKG2C is an activating NK cell receptor encoded by a gene having an unexpressed deletion variant. Cytomegalovirus (CMV) infection expands a population of NKG2C + NK cells with adaptive-like properties. Previous reports found that carriage of the deleted NKG2C - variant was more frequent in people living with HIV (PLWH) than in HIV - controls unexposed to HIV. The frequency of NKG2C + NK cells positively correlated with HIV viral load (VL) in some studies and negatively correlated with VL in others. Here, we investigated the link between NKG2C genotype and HIV susceptibility and VL set point in PLWH. NKG2C genotyping was performed on 434 PLWH and 157 HIV exposed seronegative (HESN) subjects. Comparing the distribution of the three possible NKG2C genotypes in these populations revealed that the frequency of NKG2C +/+ and NKG2C +/- carriers did not differ significantly between PLWH and HESN subjects, while that of NKG2C -/- carriers was higher in PLWH than in HESNs, in which none were found (p=0.03, χ 2 test). We were unable to replicate that carriage of at least 1 NKG2C - allele was more frequent in PLWH. Information on the pre-treatment VL set point was available for 160 NKG2C +/+ , 83 NKG2C +/- and 6 NKG2C -/- PLWH. HIV VL set point was similar between NKG2C genotypes. The frequency of NKG2C + CD3 - CD14 - CD19 - CD56 dim NK cells and the mean fluorescence intensity (MFI) of NKG2C expression on NK cells was higher on cells from CMV + PLWH who carried 2, versus 1, NKG2C + alleles. We observed no correlations between VL set point and either the frequency or the MFI of NKG2C expression. IMPORTANCE We compared NKG2C allele and genotype distributions in subjects who remained HIV uninfected despite multiple HIV exposures (HESNs) with those in PLWH. This allowed us to determine whether NKG2C genotype influenced susceptibility to HIV infection. The absence of the NKG2C -/- genotype among HESN subjects but not PLWH suggested that carriage of this genotype was associated with HIV susceptibility. We calculated the VL set point in a subset of 252 NKG2C genotyped PLWH. We observed no between-group differences in the VL set point in carriers of the three possible NKG2C genotypes. No significant correlations were seen between the frequency or MFI of NKG2C expression on NK cells with VL set point in cytomegalovirus co-infected PLWH. These findings suggested that adaptive NK cells played no role in establishing the in VL set point, a parameter that is a predictor of the rate of treatment-naïve HIV disease progression.

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Engineering lymph node homing of ex vivo–expanded human natural killer cells via trogocytosis of the chemokine receptor CCR7

Blood ◽

10.1182/blood-2011-11-389924 ◽

2012 ◽

Vol 119 (22) ◽

pp. 5164-5172 ◽

Cited By ~ 56

Author(s):

Srinivas S. Somanchi ◽

Anitha Somanchi ◽

Laurence J. N. Cooper ◽

Dean A. Lee

Keyword(s):

Lymph Node ◽

Nk Cells ◽

Natural Killer ◽

Chemokine Receptor ◽

Chemokine Receptors ◽

Nk Cell ◽

Ex Vivo ◽

Ccr7 Expression ◽

Lymph Node Homing

Natural killer (NK) cells have gained significant attention in adoptive immunotherapy for cancer. Consequently, novel methods of clinical-grade expansion of NK cells have emerged. Subsets of NK cells express a variety of chemokine receptors. However, to expand the scope of adoptively transferred NK cell homing to various malignancies, expression of corresponding chemokine receptors on NK cells is essential. Here, we have explored the use of trogocytosis as a tool to transiently express the chemokine receptor CCR7 on expanded human NK cells with the aim to enhance their homing to lymph nodes. We generated a K562-based “donor” cell line expressing CCR7, Clone9.CCR7, to transfer CCR7 onto NK cells via trogocytosis. CCR7 expression occurred in 80% of expanded NK cells within 1 hour after coculture with Clone9.CCR7. After removal of the donor cells from the coculture, the CCR7 expression on NK cells steadily declined to baseline levels by 72 hours. The acquired CCR7 receptors mediated in vitro migration of NK cells toward CCL19 and CCL21 and increased the lymph node homing by 144% in athymic nude mice. This is the first report on exploiting trogocytosis to rapidly and transiently modify lymphocytes, without direct genetic interven-tion, for adoptive transfer.

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Rituximab induces long-term remissions in patients with HIV-associated multicentric Castleman's disease

Journal of Clinical Oncology ◽

10.1200/jco.2007.25.18_suppl.8049 ◽

2007 ◽

Vol 25 (18_suppl) ◽

pp. 8049-8049

Author(s):

M. Bower ◽

T. Powles ◽

S. Williams ◽

T. Newsom-Davis ◽

M. Atkins ◽

...

Keyword(s):

Viral Load ◽

Highly Active Antiretroviral Therapy ◽

Nk Cell ◽

Treatment Options ◽

Disease Free Survival ◽

Hiv Viral Load ◽

Cd4 Cell Count ◽

Highly Active ◽

Cell Counts ◽

Anti Cd20

8049 Background: HIV associated multicentric Castlemans (MCD) is rare lympohproliferative disorder and most treatment options to date, have proved largely unsuccessful. This study investigated the efficacy of the anti CD20 monoclonal antibody rituximab in patients with this disease. Methods: Between 2003 and 2006, patients with histological proven HIV related MCD received a course of 4 infusions of rituximab 375mg/m2 at weekly intervals. Results: Twenty one consecutive patients (18 male) were recruited into this study and the median follow-up is 12 months (range 1–49). The median age was 37 years and all patients were either on highly active antiretroviral therapy (HAART) (62%) or started HAART at the time of MCD diagnosis (38%). The median CD4 cell count at MCD diagnosis was 275/mm3 (range 77–725). One died within 2 weeks of starting rituximab (not evaluable); 20 evaluable patients all achieved clinical remission of symptoms and 71% achieved a radiological response according to RECIST criteria. Rituximab lead to a significant fall in anaemia, thrombocytopenia, CRP, ESR, serum HHV8 viral load and IL-10. Rituximab caused transitory fall in the CD19 count, but had no effect on the CD4, CD8 and NK cell counts or plasma HIV viral load. The disease free survival at 2 years is 79% (95%CI: 49–100%) and overall survival is 95% (95%CI: 86–100%). Two patients have relapsed after 1.6 & 2.8 years and were successfully retreated with rituximab therapy. Conclusions: Rituximab therapy results in a sustained clinical, radiological and biochemical remission in patients with HIV related MCD. No significant financial relationships to disclose.

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Profiles of Cytotoxic Molecules In Mature NK Cell Neoplasms

Blood ◽

10.1182/blood.v116.21.4140.4140 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4140-4140

Author(s):

Nodoka Sekiguchi ◽

Naoko Asano ◽

Hideki Makishima ◽

Masanobu Momose ◽

Toshiro Ito ◽

...

Keyword(s):

Viral Load ◽

Nk Cells ◽

Liver Dysfunction ◽

Whole Blood ◽

Clinical Characteristics ◽

Nk Cell ◽

Cell Lymphoma ◽

Granzyme B ◽

Stage Iv ◽

Cytotoxic Molecules

Abstract Abstract 4140 Backgrounds: Proliferative disorders of mature natural killer (NK) cells are classified based on their pathologic and clinical characteristics and divided into three types in WHO 2008 classification, extranodal NK cell lymphoma, nasal type (ENKL), aggressive NK cell leukemia (ANKL) and chronic NK lymphocytosis (CNKL). ENKL and ANKL show strong association with EBV and are clinically aggressive, but CNKL usually dose not and takes insidious course. Cytotoxic molecules are biological important factors possibly associated with tissue injuries in NK cell neoplasms. There are several cytotoxic molecules residing in the cytotoxic granules, including granulysin; a member of saposin-like lipid binding proteins, granzyme B and perforin. However, their relevance in NK cell neoplasms is still not clear. We investigated the relationships of profiles of cytotoxic molecules with NK cell neoplasms. Materials and Methods: Patients with ENKL, ANKL and CNKL were studied. The diagnoses were made according to the WHO classification. Healthy controls and the patients with T-cell large granular lymphocyte leukemia were also included for serum granulysin evaluation. Concentration of serum granulysin was measured with ELISA. Histologic examinations of granulysin, granzyme B, perforin and TIA-1 in paraffin-embedded tissues were made by immunostaining with antibodies corresponding to the antigens. EBV viral load in the whole blood and tissues were detected by real time PCR and EBER in situ hybridization, respectively. Results and Discussion: Total 38 patients (pts) with NK cell neoplasms, including 17 ENKL, 9 ANKL, and 12 CNKL, were recruited. Median age of the patients was 42 years, 36 years, and 72 years respectively. In ENKL patients, 16 pts were nasal lymphoma and 5 pts were at stage IV. Most of the patients with ANKL showed hepatosplenomegaly, although a few CNKL pts with hepatomegaly were recognized. Liver dysfunction were observed in 29% of ENKL, 88% of ANKL, and 33% of CNKL pts. EBV was positive for all ENKL and ANKL pts, and negative for pts with CNKL. Mean serum granulylin levels were 2.52 ng/ml, 64.2 ng/ml, and 14.43 ng/ml, in ENKL, ANKL, and CNKL, respectively, (Fig.1) which were significantly higher than controls (p<0.01). ANKL pts showed the highest serum granulysin levels compared to ENKL and CNKL, and higher than those of stage IV ENKL pts (p=0.04). Serum granulysin levels are also correlated with the EBV viral load in the whole blood of EBV-positive ENKL and ANKL pts (p=0.02). After achievement of complete response, elevated serum granulysin returned to within the range of controls. Pts with higher serum granulysin tended to show liver dysfunction. Immunohistochemical studies of the cytotoxic molecules showed that all ENKL pts were positive for granzyme B and perforin. In contrast, granzyme B and perforin are positive in 57% and 85%, respectively in ANKL pts. 60% of CNKL pts were positive for granzyme B and perforin. Granulysin was positive in 50%, 29% and 25% in ENKL, ANKL and CNKL, respectively. TIA-1 was positive in all pts. From these results, three types of histologic cytotoxic molecule patterns were classified, which are granzyme B+, perforin+, granulysin+ type (n=10), granzyme B+, perforin+, granulysin-negative type (n=10), and granzyme B weakly+, perforin weakly+, granulysin-negative type (n=5). Pts in which proliferating NK cells were histologically negative for granulysin showed lower or normal serum granulysin values. Conclusions: Considerable heterogeneities were recognized in NK cell neoplasms in respect to cytotoxic molecules that are associated with certain clinical characteristics of NK cell proliferative disorders. In addition, serum granulysin might serve as a novel biomarker for NK cell lymphoma/leukemia. Disclosures: No relevant conflicts of interest to declare.

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Early CMV Replication-Associated Anti-Leukemia Effect Is Related to the Reconstitution of Specific Subsets of Natural Killer Cells after Haploidentical Stem Cell Transplantation in Acute Leukemia

Blood ◽

10.1182/blood-2018-99-115733 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2128-2128

Author(s):

Jieun Jang ◽

Haerim Chung ◽

Yu Ri Kim ◽

Hoi-kyung Jeung ◽

Ju-In Eom ◽

...

Keyword(s):

Stem Cell ◽

Viral Load ◽

Acute Leukemia ◽

Nk Cells ◽

Cell Transplantation ◽

Relapse Rate ◽

Nk Cell ◽

Cmv Infection ◽

Cmv Reactivation

Abstract Background: Cytomegalovirus (CMV) infection is a major infectious complication after allogeneic hematopoietic cell transplantation. Recently, it has been reproducibly demonstrated that CMV reactivation is associated with decreased relapse rate in patients with acute myeloid leukemia (AML). The aim of this study is to evaluate the impact of early CMV reactivation on the incidence of disease relapse in relation to the reconstitution of subsets of natural killer (NK) cells after haploidentical stem cell transplantation (haploSCT) in acute leukemia. Methods: Clinical data from 47 adult patients diagnosed with acute leukemia who underwent their first haploSCT between September 2009 and December 2017 was retrospectively analyzed. All patients were unable to find a suitable HLA-matched donor in their families or donor registries. Patient blood samples were collected prior to conditioning therapy and following haploSCT at day 30 and day 90. Peripheral blood mononuclear cells obtained from 28 patients abundant cells at every time points were analyzed for flow cytometric immunophenotyping. Expression of specific receptors (NKG2A, NKG2C, NKG2D, DNAM1 and NKp46) on the NK cells (CD56brightCD16- or CD56dim/-CD16+ cells) were serially quantified by multiparametric flow cytometry using appropriate monoclonal antibodies. Results: Median age was 38 years (range, 21-62 years), and 28 (54%) patients were male. Thirty-six (69%) patients received a transplant in their first complete remission (CR) status. Median follow-up duration was 54 months (range, 6.6-83.3 months), and all patients were CMV seropositive before receiving a transplant. Early CMV replication occurred at a median of 23 days after haploSCT in 40 of 47 patients (85%). Among these patients, 14 had more than two episodes of CMV replication throughout the follow-up period. The median peak viral load during CMV replication was 54,000 copies/mL. In univariate analysis, early CMV replication (P < 0.001), older donor age (P = 0.018), high dose of infused T cells (P = 0.022) and chronic graft-versus-host disease (GVHD, P = 0.001) were significantly associated with lower 3-year cumulative incidence of relapse after haploSCT. Early CMV replication was correlated with higher leukemia-free survival (LFS, P < 0.001). In multivariate analysis, early CMV replication (hazard ratio [HR], 0.24; 95% confidence interval [CI], 0.060 to 0.930, P = 0.039) and chronic GHVD (HR, 0.25; CI, 0.089 to 0.695; P = 0.008) were identified as independent factors for higher LFS rate. CMV reactivation was associated with higher overall survival (OS) rates and increased nonrelapse mortality, although there was no statistical significance. The viral load at the initiation of CMV-specific treatment was significantly associated with OS rates. Patients with CMV viral load higher than 45,000 copies/mL had lower OS rate compared to those with lower CMV load (34.5% versus 89.5%, P = 0.022). Longitudinal analysis of NK cell reconstitution after haploSCT showed that the CMV infection was associated with the increased expansion of CD56brightCD16dim/- NK cell, particularly in DNAM1+ NK cell subset. The rate of CD56brightCD16dim/-DNAM1+ NK cells increment was significantly higher in the patients with CMV infection compared with patients without CMV infection (P = 0.022). Importantly, we observed that the cumulative relapse rate was significantly decreased in patients with an increased CD56brightCD16dim/- DNAM1+ NK cells compared to patients with low CD56brightCD16dim/- DNAM1+ NK cells (20.4% versus 63.6% , P = 0.016). Conclusion: Early CMV replication was identified as an independent prognostic factor for LFS in acute leukemia in the haploSCT setting. Increased reconstitution of CD56brightCD16dim/- DNAM1+ NK cells was associated with CMV infection-related reduction in the relapse rate. Further studies are required to elucidate the anti-leukemia effects of these NK cell subsets associated with CMV infection in haploSCT. Disclosures Kim: Novartis Korea: Honoraria.

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Human Placental Hematopoietic Stem Cell Derived Natural Killer Cells (CYNK-001) Mediate Protection Against Influenza A Viral Infection

Blood ◽

10.1182/blood-2020-139049 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 2-3

Author(s):

Shuyang He ◽

Tanel Mahlakõiv ◽

Joseph Gleason ◽

William Van Der Touw ◽

Lin Kang ◽

...

Keyword(s):

Viral Load ◽

Lung Injury ◽

Viral Infection ◽

Nk Cells ◽

Nk Cell ◽

A549 Cells ◽

Private Company ◽

Antiviral Activities

Background: Influenza A virus (IAV) infections are associated with a high healthcare burden around the world and there is an urgent need to develop more effective therapies. Natural killer (NK) cells provide the first line of innate defense against IAV by killing infected epithelial cells, by producing antiviral cytokines and affecting adaptive immunity. Preclinical studies have demonstrated that NK cells play a pivotal role in reducing IAV-induced pulmonary infection; however, little is known about the therapeutic potential of adoptively transferred NK cells for IAV infections. Celularity Inc. is developing human placental hematopoietic stem cell-derived allogeneic, off-the-shelf NK cell therapy (CYNK-001) for the treatment of viral infections, including coronavirus disease of 2019. Here, we report the evaluation of antiviral activities of CYNK-001 against IAV infection. Methods: In vitro antiviral activities of CYNK-001 were evaluated using human alveolar epithelial cell line A549, infected with IAV strain A/PR/8/34 (H1N1) at variable multiplicity of infection (MOI). The expression of ligands for NK cell receptors was analyzed on infected A549 cells using Fc-coupled recombinant proteins. CYNK-001 was added to A549 cells 16 hours post infection. CYNK-001 degranulation was measured after 4 hours of coculture, and CYNK-001 cytotoxicity against IAV-infected A549 was measured real-time using impedance-based xCELLigence platform. In vivo antiviral and immunomodulatory activities of CYNK-001 were assessed in A/PR/8/34 (H1N1)-induced severe acute lung injury mouse model. Mice were intranasally infected with 2500 PFU IAV. PBS or 1 x 107 CYNK-001 cells were intravenously administered twice at 1 and 3 days post infection (dpi). At 6 dpi, lungs were collected for the evaluation of viral load by qPCR, lung injury and immune cell profiling by histology. Bronchoalveolar lavage fluid (BALF) was collected at 6 dpi for cytokine analysis by multiplex assays, total protein concentration by ELISA and immune cell profiling by flow cytometry. Results: In vitro, IAV infection corresponded with dose-dependent expression of ligands to NK cell-activating receptors, including NKp44, NKp46 and NKG2D. CYNK-001 cells exhibited increased IFNγ, TNFα and GM-CSF production, and elevated level of degranulation upon coculture with IAV-infected A549 cells. Cytokines in culture supernatant and CD107a expression in CYNK-001 cells were upregulated in a virus dose-dependent manner. Consistent with this finding, CYNK-001 cytotoxicity against IAV-infected A549 cells increased from 35% at 0 MOI to 50%, 60% and 75% at 0.001, 0.01 and 0.1 MOI, respectively. These data indicate that CYNK-001 cells recognize virally infected cells, resulting in specific cytotoxic elimination of the source of infection. In vivo, treatment of IAV-infected mice with CYNK-001 reduced weight loss and increased their likelihood of survival. PBS control group developed a severe disease and 37.5% mortality was observed as early as day 4. In the group treated with CYNK-001, disease onset was delayed by 2 days. qPCR analysis of viral RNA showed that CYNK-001-treated mice had lower viral load in the lung than vehicle-treated mice, demonstrating antiviral function of CYNK-001 in vivo. CYNK-001-treated mice had reduced lung injury as assessed by lower total protein concentration in BALF. Moreover, CYNK-001 reduced BALF murine cytokines and chemokines, including IFNγ (p<0.001), IL-6, TNFα, MCP-1 (p<0.05), CXCL2 and CXCL9. Lastly, immunohistochemical analysis of the lung showed that CYNK-001-treated mice had an altered immune response to IAV with higher number of CD68+ macrophages and CD8+ T cells at 6 dpi. Conclusions: Our in vitro and in vivo data show the promising antiviral activities of CYNK-001 against IAV infection. In a severe IAV infection mouse model, CYNK-001 treatment demonstrates lower mortality rate, lower weight loss, lower lung viral load and reduced lung injury along with reduced inflammation. These results support our hypothesis that the adoptive transfer of CYNK-001 could reduce the burden of viral infection through the elimination of infected epithelial cells, coordinate a more effective immune response, and result in a clinical benefit in patients with severe viral infection. Disclosures He: Celularity Inc.: Current Employment. Mahlakõiv:Celularity Inc.: Current Employment. Gleason:Celularity Inc.: Current Employment, Current equity holder in private company. Van Der Touw:Celularity Inc.: Current Employment. Kang:Celularity Inc.: Current Employment. Hariri:Celularity Inc.: Current Employment, Current equity holder in private company. Zhang:Celularity Inc.: Current Employment, Current equity holder in private company.

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