The longest persistence of viable SARS-CoV-2 with recurrence of viremia and relapsing symptomatic COVID-19 in an immunocompromised patient – a case study

Abstract Background Immunocompromised patients show prolonged shedding of SARS-CoV-2 in nasopharyngeal swabs. We report a case of a prolonged persistence of viable SARS-CoV-2 associated with clinical relapses of COVID-19 in a patient with mantle-cell lymphoma who underwent treatment with R-BAC (rituximab, bendamustine, cytarabine) with consequent lymphopenia and hypogammaglobulinemia. Methods Nasopharyngeal swabs and blood samples were tested for SARS-CoV-2 by Real time-PCR (RT-PCR). On five positive nasopharyngeal swabs, we performed viral culture and next generation sequencing. We analysed the patients’ adaptive and innate immunity to characterize T and NK cell subsets. Results SARS-CoV-2 RT-PCR on nasopharyngeal swabs samples remained positive for 268 days. All five performed viral cultures were positive and genomic analysis confirmed a persistent infection with the same strain. Viremia resulted positive in three out of four COVID-19 clinical relapses and cleared each time after remdesivir treatment. T and NK cells dynamic was different in aviremic and viremic samples and no SARS-CoV-2 specific antibodies were detected throughout the disease course. Conclusions In our patient, SARS-CoV-2 persisted with proven infectivity for over eight months. Viremia was associated with COVID-19 relapses and remdesivir treatment was effective in viremia clearance and symptoms remission, although it was unable to clear the virus from the upper respiratory airways. During the viremic phase, we observed a low frequency of terminal effector CD8+ T lymphocytes in peripheral blood that are probably recruited in inflammatory tissue for viral eradication. In addition we found a high level of NK cells repertoire perturbation with a relevant involvement during SARS-CoV-2 viremia.

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The longest persistence of viable SARS-CoV-2 with recurrence of viremia and relapsing symptomatic COVID-19 in an immunocompromised patient – a case study

10.1101/2021.01.23.21249554 ◽

2021 ◽

Author(s):

Chiara Sepulcri ◽

Chiara Dentone ◽

Malgorzata Mikulska ◽

Bianca Bruzzone ◽

Alessia Lai ◽

...

Keyword(s):

Nk Cells ◽

Nk Cell ◽

Low Frequency ◽

Genomic Analysis ◽

Rt Pcr ◽

Mean Values ◽

Viral Culture ◽

Viremic Phase ◽

High Level

AbstractBackgroundImmunocompromised patients show prolonged shedding of SARS-CoV-2 in nasopharyngeal swabs. We report a case of a prolonged persistence of viable SARS-CoV-2 associated with clinical relapses of COVID-19 in a lymphoma patient.MethodsNasopharyngeal swabs and blood samples were tested for SARS-CoV-2 by Real time-PCR (RT-PCR). On five positive nasopharyngeal swabs, we performed viral culture and next generation sequencing. We analysed the patients’ adaptive and innate immunity to characterize T and NK cell subsets.FindingsSARS-CoV-2 RT-PCR on nasopharyngeal swabs samples remained positive with cycle threshold mean values of 22 ± 1·3 for over 8 months. All five performed viral cultures were positive and genomic analysis confirmed a persistent infection with the same strain. Viremia resulted positive in three out of four COVID-19 clinical relapses and cleared each time after remdesivir treatment. T and NK cells dynamic was different in aviremic and viremic samples and no SARS-CoV-2 specific antibodies were detected throughout the disease course.InterpretationIn our patient, SARS-CoV-2 persisted with proven infectivity for over eight months. Viremia was associated with COVID-19 relapses and remdesivir treatment was effective in viremia clearance and symptoms remission, although it was unable to clear the virus from the upper respiratory airways. During the viremic phase, we observed a low frequency of terminal effector CD8+ T lymphocytes in peripheral blood that are probably recruited in inflammatory tissue for viral eradication. In addition we found a high level of NK cells repertoire perturbation with a relevant involvement during SARS-CoV-2 viremia.FundingNone.

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Decrease post-transplant relapse using donor-derived expanded NK-cells

Leukemia ◽

10.1038/s41375-021-01349-4 ◽

2021 ◽

Author(s):

Stefan O. Ciurea ◽

Piyanuch Kongtim ◽

Doris Soebbing ◽

Prashant Trikha ◽

Gregory Behbehani ◽

...

Keyword(s):

Nk Cells ◽

Nk Cell ◽

Ex Vivo ◽

Disease Free Survival ◽

Dependent Manner ◽

Post Transplant ◽

Haploidentical Transplantation ◽

High Doses ◽

High Level

AbstractIn this phase I/II clinical trial, we investigated the safety and efficacy of high doses of mb-IL21 ex vivo expanded donor-derived NK cells to decrease relapse in 25 patients with myeloid malignancies receiving haploidentical stem-cell transplantation (HSCT). Three doses of donor NK cells (1 × 105–1 × 108 cells/kg/dose) were administered on days −2, +7, and +28. Results were compared with an independent contemporaneously treated case-matched cohort of 160 patients from the CIBMTR database.After a median follow-up of 24 months, the 2-year relapse rate was 4% vs. 38% (p = 0.014), and disease-free survival (DFS) was 66% vs. 44% (p = 0.1) in the cases and controls, respectively. Only one relapse occurred in the study group, in a patient with the high level of donor-specific anti-HLA antibodies (DSA) presented before transplantation. The 2-year relapse and DFS in patients without DSA was 0% vs. 40% and 72% vs. 44%, respectively with HR for DFS in controls of 2.64 (p = 0.029). NK cells in recipient blood were increased at day +30 in a dose-dependent manner compared with historical controls, and had a proliferating, mature, highly cytotoxic, NKG2C+/KIR+ phenotype.Administration of donor-derived expanded NK cells after haploidentical transplantation was safe, associated with NK cell-dominant immune reconstitution early post-transplant, preserved T-cell reconstitution, and improved relapse and DFS. TRIAL REGISTRATION: NCT01904136 (https://clinicaltrials.gov/ct2/show/NCT01904136).

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Extent of Cytomegalovirus Replication in the Human Host Depends on Variations of the HLA-E/UL40 Axis

mBio ◽

10.1128/mbio.02996-20 ◽

2021 ◽

Vol 12 (2) ◽

Cited By ~ 1

Author(s):

Hannes Vietzen ◽

Timo Rückert ◽

Svenja Hartenberger ◽

Claudia Honsig ◽

Peter Jaksch ◽

...

Keyword(s):

Lung Transplantation ◽

Nk Cells ◽

Human Cytomegalovirus ◽

Cell Response ◽

Nk Cell ◽

Content Type ◽

Immunosuppressed Patients ◽

Hcmv Replication ◽

Type Gene ◽

High Level

ABSTRACT Human cytomegalovirus (HCMV) may cause severe infections in lung transplant recipients (LTRs). In response to HCMV infections, a subset of NKG2C+ NK cells expands, which limits HCMV replication and is characterized by high expression of the activating NKG2C/CD94 and absence of the inhibitory NKG2A/CD94 receptor. Both receptors bind to HLA-E, which is stabilized by HCMV-encoded UL40 peptides. HLA-E and UL40 occur as different genetic variants. In this study, we investigated the interplay between the human NK cell response and the infecting HCMV-UL40 strain, and we assessed the impact of HCMV-UL40 and of donor- and recipient-encoded HLA-E*0101/0103 variants on HCMV replication after lung transplantation. We included 137 LTRs displaying either no or low- or high-level (>1,000 copies/ml plasma) viremia. HCMV-UL40 and HLA-E*0101/0103 variants were determined. UL40 diversity was investigated by next-generation sequencing. UL40 peptide-dependent NK cell cytotoxicity was assessed by flow cytometry. Donor-encoded HLA-E*0101/0103 was significantly associated with development of high-level viremia after transplantation (P = 0.007). The HCMV-UL40 variant VMAPRTLIL occurred significantly more frequently in highly viremic LTRs, and the variant VMTPRTLIL occurred significantly more frequently in low-viremic LTRs (P = 0.004). This difference was associated with a better inhibition of NKG2A+ NKG2C− NK cells by VMAPRTLIL (P < 0.001). In LTRs with repeated high-level viremic episodes, HCMV strains with UL40 variants displaying low affinity to the patients’ HLA-E variant emerged over time. The HLA-E-UL40 axis has a substantial impact on the level of HCMV replication in LTRs. The interplay between UL40 peptide variants, the recipient HLA-E status, and the activation of inhibitory NKG2A+ NKG2C− cells is of major importance for development of high-level viremia after lung transplantation. IMPORTANCE Infection with human cytomegalovirus (HCMV) is associated with substantial morbidity in immunosuppressed patients and after congenital infections. Therefore, development of a vaccine against HCMV is a main public health priority. Revealing the complex interaction between HCMV and host responses, is of utmost importance for understanding viral pathogenesis and for vaccine design. The present data contribute to the understanding of HCMV-specific host immune responses and reveal specifically the interaction between HLA-E and the virus-encoded UL40 peptide, which further leads to a potent NK cell response. We demonstrate that this interaction is a key factor for reduction of virus replication in immunosuppressed patients. We further show that distinct naturally occurring HCMV-UL40 variants reduce the activation of a specific subpopulation of host NK cells and thereby are associated with high-level viremia in the patients. These findings will allow the characterization of patients at risk for severe HCMV infection and contribute to strategies for HCMV vaccine development.

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A Case of Hypersensitivity to Mosquito Bites with Type III Latency of EB Virus Infection in NK Cells with Prolonged Survival Due to Induction of Specific CD8+ Cytotoxic T Cells.

Blood ◽

10.1182/blood.v108.11.1263.1263 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1263-1263

Author(s):

Shigeru Tsuchiya ◽

Wei Du ◽

Hideaki Kikuta ◽

Toru Uchiyama ◽

Masaei Onuma ◽

...

Keyword(s):

Nk Cells ◽

Mononuclear Cells ◽

Nk Cell ◽

Pcr Analysis ◽

Rt Pcr ◽

Type Iii ◽

Ebv Infection ◽

Eb Virus ◽

Promoter Usage ◽

Methylation Patterns

Abstract Hypersensitivity to mosquito bites (HMB) is a rare disease classified into chronic active EB Virus (EBV) infection (CAEBV), characterized by clonal expansion of EBV-infected NK cells. The immunological background of the disease has not been elucidated yet. In this report we extensively studied EBV infected NK cells of a patient with HMB in view of several aspects, including latency, usage of promoter Cp/Wp and Qp of EBNAs, and induction of CD8+ cytotoxic T lymphocytes (CTL) to peptide antigens in order to know the relation between latency of EBV infected NK cells and induction of CTL. Case report: A female patient aged 19 complained of skin symptoms after a mosquito bite. A blister developed at the bite site that subsequently transformed into ulcer. The patient also had general fever and liver dysfunction. Past medical history showed that she suffered from HMB when she was 7 years old. The titers of serum antibodies to EBV were as follows: viral capsid antigen (VCA) IgG, 1280X; IgA, 80X; IgM, 10X; EBNA, 20X; EA DR IgG, 160X and IgA, 10X. These data strongly indicated lytic type of EBV infection. Subset analysis of peripheral blood lymphocytes revealed CD3+ lymphocytes, 50.2%; CD4+, 34.2%; CD8+, 12.7%; CD19, 10.0% and CD56/16+, 22.6%. Real time polymerase chain analysis of her peripheral mononuclear cells showed markedly increased copy number of EBV genome (5 x 10^5 copies/μgDNA). Purification of each lymphocyte fraction by immunomagnetic beads and subsequent PCR analysis showed that EBV infected lymphocytes were NK cells. Latency and Cp and Qp of EBNA promoter usage analyses: In order to determine the latency of EBV infected NK cells RT-PCR was carried out. EBNA1, EBNA2, LMP1, LMP2A, and EBER1 were positive and BZLF was negative. These results indicated that EBV infection in NK cells is associated with type III latency. RT-PCR analyses confirmed that both promoters Cp/Wp and Qp were involved in this disease. Through bisulfite PCR analysis, eighty four % of Cp was found to be methylated, on the other hand, only 6% of Qp was methylated. These methylation patterns were similar to those of hemophagocytic syndrome with latency III, also known in a few patients with CAEBV. Detection and expansion of EBV specific CD8+ CTL: We used HLA-A24 restricted BRLF1, EBNA3A, EBNA3B and LMP2 tetramers for quantification of specific CTL and we found 0.48% of BRLF1 and 0.15% of EBNA3 peptide-specific CTL among CD8+ cells. These cells could be expanded to 6.6% for BRLF1 and 0.22% for EBNA3A peptide-specific CTL after stimulation with an autologous lymphoblastoid cell line (LCL) for 10 days and then with LCL and IL-2 for 7 days. Conclusion: In this study we reported a long-term survivor of HMB with EBV infected NK cell proliferation. Promoter usage of EBNA genes and their methylation patterns might be important to determine latency and host immune responses against EBV infected cells.

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CD137 Expression Is Enhanced in EBV-Infected T or NK Cells by Viral Protein LMP1 and Mediates Anti-Apoptotic Intracellular Signaling through NF-ĸB Activation.

Blood ◽

10.1182/blood.v114.22.1929.1929 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1929-1929

Author(s):

Ayako Arai ◽

Ken-ichi Imadome ◽

Mayumi Takahashi ◽

Koichi Naka ◽

Tetsuya Fukuda ◽

...

Keyword(s):

Cell Surface ◽

Nk Cells ◽

Intracellular Signaling ◽

Mononuclear Cells ◽

Nk Cell ◽

Jurkat Cells ◽

Rt Pcr ◽

Infected Cells ◽

Ebv Dna

Abstract Abstract 1929 Poster Board I-952 Epstein-Barr virus (EBV) can infect not only B cells but also T or NK cells uncommonly and causes lymphoid malignancies, such as extranodal NK/T-cell lymphoma nasal type (ENKL), aggressive NK-cell leukemia, and EBV-positive T/NK-cell lymphoproliferative disease (EBV-T/NK-LPD), which is also known as chronic active EBV infection. However, why and how EBV infects T or NK cells and the mechanism of action responsible for these EBV-induced malignancies have not been elucidated to date. To clarify the molecular mechanism underlying development of EBV-T/NK-LPD, we focused on costimulatory receptor CD137, which is expressed on the surface of activated T cells and plays a pivotal role in their proliferation, survival, and differentiation. We investigated CD137 expression on the surface of EBV-infected T/NK cells (EB-T/NK cells) by flow cytometry. First, three EBV-positive T and NK cell lines, SNT8, SNK6, and SNT16, were obtained for examination. These cell lines had been established from primary lesions of ENKL patients (SNT8 and SNK6) and peripheral blood of an EBV-T/NK-LPD patient (SNT16). CD137 expression was confirmed on the cell surface of these cells, whereas the EBV-negative T and NK cell line, Jurkat and KHYG1 cells, respectively, were negative for CD137. Next, we investigated expression on the surface of EB-T/NK cells derived from EBV-T/NK-LPD patients. EBV-T/NK-LPD was diagnosed according to the following criteria: presence of persistent infectious mononucleosis-like symptoms, elevation of EBV-DNA titer in the peripheral blood (PB), and detection of EBV-infected T or NK cells. To detect the infected cells, we isolated peripheral mononuclear cells and divided them into CD19-, CD4-, CD8-, or CD56-positive fractions using antibody-conjugated magnetic beads. Next, we measured the EBV-DNA titer of each fraction by quantitative RT-PCR. Nine patients (aged 8–41 years; 4 male, 5 female; 4 T and 5 NK cell types) were diagnosed with EBV-T/NK-LPD. Then, we examined surface CD137 expression of the infected cells of each patient. Expression was detected in 7 of 9 patients. Control cells (PB mononuclear cells of a healthy donor, who was negative for EBV-DNA titer in the PB) did not express the molecule. We also examined transcription of CD137 mRNA by RT-PCR assay and detected it in all the 12 EB-T/NK-cell samples described above. From these results we concluded that CD137 expression was induced at the level of both mRNA and protein in EB-T/NK cells. To investigate the molecular mechanism of CD137 overexpression in EBV-T/NK cells, we examined the influence of viral proteins on CD137 expression. EB-T/NK cells express EBV-encoded proteins, including LMP1, LMP2A, LMP2B, and EBNA1 (latency type 2). We cotransfected expression plasmids for these proteins with a luciferase reporter plasmid containing the CD137 gene promoter in Jurkat cells and performed a luciferase assay. LMP1 significantly upregulated the CD137 promoter activity, although the other molecules did not. Furthermore, in a transient expression assay of these viral proteins using Jurkat cells, transcription of endogenous mRNA of CD137 was upregulated only in the LMP1 transfectant. These results indicate that LMP1 may transactivate CD137 transcription and expression in EBV-T/NK cells. Next, we investigated the role of CD137 in developing EBV-T/NK-LPD. We cultured the above-mentioned CD137-expressing EBV-T/NK cells on CHO cells that stably express human CD137L on the cell surface. NF-ĸB activation was detected in CD137-positive EBV-T/NK cells that were cocultured with CD137L-expressing CHO cells. We confirmed that both p50 and p52 translocated to the nucleus, indicating that both canonical and non-canonical pathways for NF-ĸB activation were activated downstream of CD137. Finally, we investigated the role of CD137-mediated NF-ĸB activation in the development of EBV-T/NK-LPD. We cocultured EB-T/NK cells on CHO-wt or CHO-CD137L with VP-16 for 48 h and determined apoptosis by measuring DiCO6 uptake. We noted that stimulation of CD137 significantly suppressed VP-16-induced apoptosis of these cells. Together, these results indicate that EBV-infected T/NK-cells express CD137 on the cell surface, which may be induced by LMP1 and activate the anti-apoptotic intracellular signaling pathway through NF-ĸB activation. This pathway may contribute to immortalization of the infected cells and development of EBV-T/NK-LPD. Disclosures: No relevant conflicts of interest to declare.

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Micrornas Promote Activation and Maturation of Natural Killer Cells Through Toll-Like Receptor Signaling.

Blood ◽

10.1182/blood.v120.21.2160.2160 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2160-2160 ◽

Cited By ~ 1

Author(s):

Jianhua Yu ◽

Shun He ◽

Lai-Chu Wu ◽

Hsiaoyin Mao ◽

Sumithira Vasu ◽

...

Keyword(s):

Nk Cells ◽

Real Time ◽

Cell Activation ◽

Nk Cell ◽

Cell Maturation ◽

Toll Like Receptor ◽

Rt Pcr ◽

Tlr Signaling ◽

Healthy Donors

Abstract Abstract 2160 Introduction: MicroRNAs (miRNAs) are short ribonucleic acids, which consist of an average of 22 nucleotides, and bind to complementary sequences of target mRNAs to result in translational repression or target degradation, thus silencing gene expression. MiRNAs can be abundantly found in circulating blood, yet whether, as a class of regulatory molecules, they may interact with innate immune natural killer (NK) cells has not been explored. Methods: Human NK cells were first enriched from the peripheral blood of healthy donors by negative selection using RosetteSep NK cell enrichment cocktail, followed by positive selection using anti-CD56 microbeads. After purity of ≥ 99% was confirmed by flow cytometry, NK cells were used for experiments. After being isolated from healthy donor serum by ExoQuick Exosome precipitation and verified by immunoblotting for CD9 expression, exosomes were assessed for miRNA content via real-time reverse-transcriptase (RT)-PCR using TaqMan miRNA assays. Purified NK cells were stimulated with either whole exosomes or miRNAs complexed with DOTAP, a liposomal transfection reagent. Downstream activation of Toll-like receptor (TLR) signaling by miRNAs was measured via immunoblotting for NF-kB, and its inhibition was similarly assayed in the presence of TLR blocking antibodies. Flow cytometry was used to assess NK cell activation (via CD69 surface expression) and NK cytotoxicity against tumor cells (in a CD107a degranulation assay). IFN-g production was measured via Real-time RT-PCR and enzyme-linked immunosorbent assay (ELISA). For in vivo stimulation, a complex consisting of miRNAs and Lipofectamine 2000 was administered by tail-vein injection. NK cell activation was then measured using the aforementioned in vitro assays. After in vivo stimulation with miRNAs, which was performed in the presence of NK cells or following NK depletion by TM-β1 (IL-2/15Rβ) mAb, development of implanted lymphoma tumor cells was monitored by bioluminescent imaging. NK cells purified from lymphoma patients and from healthy donors were assessed for expression of the NF-kB signaling component, p65, and TLRs via real-time RT-PCR. NK cell maturation was analyzed by flow cytometric staining for surface receptors, such as CD56 and CD94, indicative of NK cell maturation. Results: We found that, in the presence of a low dose IL-12, treatment of human NK cells with several mature miRNAs induced CD69 expression, IFN-g production, and expression of the degranulation marker, CD107a. MiRNA-containing exosomes freshly isolated from normal human donors were also able to activate NK cells, even in the absence of IL-12. In vivo, infusion of several miRNAs into the peripheral blood similarly activated murine NK cells, while T cells were not activated. Furthermore, miRNA administration significantly protected mice from developing tumors, and this occurred in an NK cell-dependent manner. Interestingly, miRNAs also augmented expression of surface markers associated with NK cell maturation, such as CD56 and CD94, suggesting that miRNAs may play a role in promoting NK cell maturation. Mechanistically, we found that stimulation with miRNAs led to downstream activation of NF-kB. This effect was blunted upon blockade of TLR (e.g. TLR1) signaling, and was attenuated in lymphoma patients. Conclusion: Collectively, we provide the first evidence that extrinsic miRNAs, as a class of regulatory molecules, directly activate and may also promote the maturation of NK cells. These effects on NK cell activation and maturation are mediated, at least in part, by the TLR signaling pathway. This phenomenon may be important for normal host defense against infection and/or malignant transformation. Our studies indentify a new function of miRNAs with physiological relevance, and their potential for applications in preventing or treating cancer and infections either alone or as an adjuvant. Disclosures: Jaglowski: Pharmacyclics: Research Funding.

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Interleukin-15 Enhances Cytotoxicity, Receptor Expression, and Expansion of Neonatal Natural Killer Cells in Long-Term Culture

Clinical and Diagnostic Laboratory Immunology ◽

10.1128/cdli.11.5.879-888.2004 ◽

2004 ◽

Vol 11 (5) ◽

pp. 879-888 ◽

Cited By ~ 22

Author(s):

Sunwoong S. Choi ◽

Vaninder S. Chhabra ◽

Quoc H. Nguyen ◽

Bonnie J. Ank ◽

E. Richard Stiehm ◽

...

Keyword(s):

Nk Cells ◽

Natural Killer ◽

Mononuclear Cells ◽

Cell Function ◽

Nk Cell ◽

Newborn Infants ◽

Receptor Expression ◽

Developmental Defects ◽

Interleukin 15 ◽

High Level

ABSTRACT Newborn infants have a higher susceptibility to various pathogens due to developmental defects in their host defense system, including deficient natural killer (NK) cell function. In this study, the effects of interleukin-15 (IL-15) on neonatal NK cells was examined for up to 12 weeks in culture. The cytotoxicity of fresh neonatal mononuclear cells (MNC) as assayed by K562 cell killing is initially much less than that of adult MNC but increases more than eightfold after 2 weeks of culture with IL-15 to a level equivalent to that of adult cells. This high level of cytotoxicity was maintained for up to 12 weeks. In antibody-dependent cellular cytotoxicity (ADCC) assays using CEM cells coated with human immunodeficiency virus gp120 antigen, IL-15 greatly increased ADCC lysis by MNC from cord blood. IL-15 increased expression of the CD16+ CD56+ NK markers of cord MNC fivefold after 5 weeks of incubation. Cultures of neonatal MNC with IL-15 for up to 10 weeks resulted in a unique population of CD3− CD8+ CD56+ cells (more than 60%), which are not present in fresh cord MNC. These results show that IL-15 can stimulate neonatal NK cells and sustain their function for several weeks, which has implications for the clinical use of IL-15.

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The Mechanism of Activated TGF-β1 Inhibiting GVL Effects of Bone Marrow NK Cells Leading to Early Relapse after Transplantation

Blood ◽

10.1182/blood-2020-143355 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 48-49

Author(s):

Dongyao Wang ◽

Xiaoyu Zhu ◽

Zimin Sun

Keyword(s):

Bone Marrow ◽

Nk Cells ◽

Transforming Growth Factor ◽

Immune Escape ◽

Nk Cell ◽

Conflicts Of Interest ◽

Transforming Growth Factor Β1 ◽

Hematopoietic Stem ◽

High Level ◽

Tgf Β1

Allogeneic hematopoietic stem-cell transplantation (allo-HSCT) is one of the best ways to cure acute myeloid leukemia (AML). However, patients with AML undergoing allo-HSCT are at risk of relapse, the mechanism remains poorly understood. Here, we demonstrated the significant decrease of nature killer (NK) cells, and the declined proportion of multi-functional effector NK cells in bone marrow from patients who had a relapse in 3 months after allo-HSCT. Furthermore, we verified the levels of activated Transforming growth factor-β1 (TGF-β1), not the total TGF-β1, increased in bone marrow of these patients. This high level activated TGF-β1 was correlated with reduced cytotoxicity of NK cell, and contributed to immune escape of tumor cells. Moreover, the expression of glycoprotein A repetitions predominant (GARP), which is critical to TGF-β1 activation, high expressed in CD4+ T cells of patients who had a relapse. These data reveal a mechanism of immune escape and proposes approaches for therapeutic administration of NK cells in order to reverse suppression of activated TGF-β1 during early allo-HSCT. Disclosures No relevant conflicts of interest to declare.

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STAT5A Overexpression Enhances Killer Immunoglobulin Receptor (KIR) Expression in Developing NK Cells and Is Associated with a Loss of Reverse Transcription from the Proximal KIR Promoter.

Blood ◽

10.1182/blood.v110.11.798.798 ◽

2007 ◽

Vol 110 (11) ◽

pp. 798-798

Author(s):

Frank M. Cichocki ◽

Todd Lenvik ◽

Stephen K. Anderson ◽

Jeffrey S. Miller

Keyword(s):

Dna Methylation ◽

Stem Cell ◽

Nk Cells ◽

Reverse Transcription ◽

Nk Cell ◽

Hla Class I ◽

Class I ◽

Proximal Promoter ◽

Rt Pcr ◽

Expression Levels

Abstract Down-regulation of HLA-class I molecules is commonly observed in virally infected and malignantly transformed cells and can contribute to the ability of these cells to escape recognition by the adaptive immune system. NK cells are able to detect the loss of even single HLA alleles on potential target cells through their clonally distributed HLA-class I-specific inhibitory receptors (KIR). While it is well established that individual KIR gene expression is strongly correlated with the DNA methylation status of CpG dinucleotides in areas surrounding the transcription initiation region, the mechanisms that regulate variegated KIR expression are largely unknown. The manipulation of KIR expression is of considerable interest due to the widely reported correlation between KIR ligand status and patient survival in hematopoietic stem cell transplant settings. Because the transcription factor STAT5 is activated downstream of the BCR/ABL oncogene, which we have previously shown to substantially augment KIR expression levels in primary NK cells, we hypothesized that STAT5 could directly influence KIR expression. To test this hypothesis, we purified CD34+ hematopoietic progenitor cells from umbilical cord blood and retrovirally transduced these cells with either a control murine stem cell virus (MSCV) vector encoding eGFP alone or an MSCV vector encoding both eGFP and a constitutively active form of STAT5A, termed STAT5A 1*6. CD34/eGFP-positive cells were then purified and cultured on the EL08-D1 stroma line, known to support NK cell development. After a period of 28 days in culture, 6.25±2.73% of the NK cells in eGFP control cultures were KIR-positive compared with 29.3±4.27% of STAT5A 1*6 transductants. To more closely examine individual KIR expression at the transcriptional level, we carried out a quantitative RT-PCR analysis with probe sets previously validated for amplification of individual KIR (Cooley et al., Blood). We observed a general increase in the mRNA expression levels of both inhibitory and activating KIR in STAT5A 1*6-transduced cells. In order to investigate the mechanism underlying the increased KIR expression observed in the STAT5A 1*6-transductants, we performed an RT-PCR specific for the reverse transcript originating from the proximal promoter of the KIR3DL1 gene. In a model proposed by Anderson et al., reverse transcription from the bidirectional proximal promoter of variegated KIR genes may be responsible for the silencing of the KIR locus through the production of dsRNA, which then induces DNA methylation in a siRNA-dependent manner. Our RT-PCR results from multiple donors clearly show an absence of reverse proximal transcript in cells transduced with the STAT5A 1*6 construct in contrast to high levels of transcript in eGFP controls. These results provide evidence for a role of STAT5A in the induction of KIR expression and support a model of KIR regulation involving intergenic transcription and possibly siRNA-mediated gene silencing. A better understanding of how to manipulate these KIR expression patterns may be of benefit to exploit the potential of NK cell therapy to improve transplant outcomes.

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Lenalidomide Exhibits Activity in Mantle Cell Lymphoma through Increased NK Cell Mediated Cytotoxicity

Blood ◽

10.1182/blood.v126.23.821.821 ◽

2015 ◽

Vol 126 (23) ◽

pp. 821-821 ◽

Cited By ~ 1

Author(s):

Patrick Hagner ◽

Hsiling Chiu ◽

Maria Ortiz-Estevez ◽

Tsvetan Biyukov ◽

Carrie Brachman ◽

...

Keyword(s):

T Cells ◽

Nk Cells ◽

Cell Lines ◽

Research Funding ◽

Nk Cell ◽

Employment Equity ◽

Mantle Cell ◽

Apoptosis Assay ◽

Equity Ownership

Abstract Introduction: Lenalidomide (Len) is indicated for the treatment of relapsed/refractory (R/R) Mantle Cell Lymphoma (MCL) in the United States and Switzerland. Len binds to the cullin 4 ring E3 ubiquitin ligase complex resulting in ubiquitination and subsequent proteasomal degradation of lymphoid transcription factors Aiolos and Ikaros leading to stimulation of immune cells, such as T-cells. Clinical trial CC-5013-MCL-002 (NCT00875667) is a randomized open-label phase II study in R/R MCL patients in which Len was given orally at 25 mg/day on days 1-21 of each 28-day cycle until progression (N=170). The control arm consisted of investigator choice of single-agent rituximab, gemcitabine, fludarabine, chlorambucil, or cytarabine (N=84). We explored the immune effects of Len treatment in MCL patients enrolled in CC-5013-MCL-002 and further investigated our findings in in vitro MCL co-culture models. Methods: Peripheral blood samples for exploratory analysis were collected at Cycle 1 Day 1 (C1D1, pre-treatment), Cycle 1 Day 4 (C1D4), Cycle 2 Day 15 (C2D15) and at treatment discontinuation. Flow cytometric profiling of T, B and natural killer (NK) cell subsets was performed and differences were analyzed for correlation with clinical outcomes (response rate and progression free survival [PFS]). Cell dependent cytotoxicity was measured in 1) anti-CD3 stimulated peripheral blood mononuclear cells (PBMC) treated with vehicle or 1-10000 nM Len for 3 days and incubated with target tumor cells for an additional 4 hours followed by an apoptosis assay as measured by Annexin V/ToPro-3 flow cytometry and 2) negatively selected CD56+ NK cells stimulated with IL-2 and treated with Len (1 nM to 10 μM) for 18 hrs and incubated with target tumor cells for an additional 4 hours followed by apoptosis assay. Results: At baseline, no significant differences were observed in the absolute levels of immune subsets when comparing non-responders (NR) and responders (R) in either Len (NR=11, R=23) or control (NR=4, R=5) arms. However, in the Len arm, significantly elevated (adj. p < 0.05) proportions of CD3-CD56+CD16+ NK cells (difference of means = 8.73; 95%CI [4.48, 12.98]) were observed at C1D4 compared to baseline in the R (N=19) outcome sub-group compared to NR (N=11). A similar trend in levels of NK subsets was observed at C2D15, however the difference was not significant. In addition, elevated proportions of CD3-CD56+CD16+ NK cells (p≤0.016) at C1D4 relative to total lymphocytes correlated significantly to longer PFS in the Len arm. Immune subset analysis in the control arm did not show any correlation to response or PFS at any visit. The mechanism whereby NK cell modulation contributes to clinical benefit demonstrated by Len in patients was further explored in in vitro co-culture systems with MCL cell lines. Len treated PBMC co-cultured with Jeko-1, Granta-519, and Mino MCL cell lines resulted in 38-47.5% more apoptosis compared to DMSO (p≤0.001). We examined the effect of Len on Aiolos and Ikaros protein expression in CD56+ NK and CD3+ T cells within anti-CD3 antibody stimulated PBMCs treated with DMSO or various concentrations of Len (1 nM to 10 μM) for 72 hours. Degradation of both Aiolos (40%) and Ikaros (95%) was observed after drug treatment in CD56+ NK cells. Aiolos and Ikaros levels were also monitored in CD3+ T cells and showed decreased levels after Len treatment, consistent with previous reports (Gandhi, 2014; Kronke, 2014). Furthermore, purified CD56+ NK cell mediated cytotoxicity produced a similar pro-apoptotic effect as the PBMC assay in all MCL cell lines versus DMSO (p≤0.01). Supernatants from co-cultures of NK cells with MCL cell lines showed significantly elevated granzyme B levels as compared to DMSO controls (p≤0.0001), suggesting that the apoptotic effects observed are induced by granzyme B. Conclusions: Lenalidomide is an immune modulating agent and NK cell modulation in particular may play a role in its clinical activity in MCL. A significant increase in proportions of NK cell subsets (vs total lymphocytes) at C1D4 versus baseline was observed and is a potential response indicator of favorable clinical outcome in R/R MCL patients treated with Len. In vitro, Len enhances cell mediated cytotoxicity of MCL cell lines in two co-culture model systems. Understanding NK cell mediated mechanism(s) has potential to enhance guiding patient selection strategies and rational combination therapies of lenalidomide in MCL. Disclosures Hagner: Celgene: Employment, Equity Ownership. Chiu:Celgene: Employment, Equity Ownership. Ortiz-Estevez:Celgene: Employment, Equity Ownership. Biyukov:Celgene: Employment, Equity Ownership. Brachman:Celgene: Employment, Equity Ownership. Trneny:Celgene: Consultancy, Honoraria, Other: Travel, accommodations, expenses, Research Funding. Morschhauser:Genentech Inc./Roche: Other: Advisory boards. Stilgenbauer:AbbVie, Amgen, Boehringer-Ingelheim, Celgene, Genentech, Genzyme, Gilead, GSK, Janssen, Mundipharma, Novartis, Pharmacyclics, Roche: Consultancy, Honoraria, Research Funding. Milpied:Celgene: Honoraria, Research Funding. Musto:Sandoz: Consultancy; Celgene: Honoraria; Roche: Honoraria; Sanofi: Consultancy; Genzyme: Consultancy; Novartis: Honoraria; Janssen: Honoraria; Mundipharma: Honoraria. Martinelli:AMGEN: Consultancy; Ariad: Consultancy; Pfizer: Consultancy; ROCHE: Consultancy; BMS: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; MSD: Consultancy. Heise:Celgene: Employment, Equity Ownership. Daniel:Celgene: Employment, Equity Ownership. Chopra:Celgene: Employment, Equity Ownership. Carmichael:Celgene: Employment, Equity Ownership. Trotter:Celgene Corporation: Employment. Gandhi:Celgene: Employment, Equity Ownership. Thakurta:Celgene Corporation: Employment, Equity Ownership.

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