Obesity modulates NK-cell activity via LDL & DUSP1 signaling for Populations with Adverse Social Determinants.

African American (AA) women are disproportionally affected by obesity and hyperlipidemia, particularly in the setting of adverse social determinants of health (aSDoH) contributing to health disparities. Obesity, hyperlipidemia, and aSDoH appear to impair Natural Killer cells (NKs). As potential common underlying mechanisms are largely unknown, we sought to investigate common signaling pathways involved in NK dysfunction related to obesity and hyperlipidemia in AA women from under-resourced neighborhoods.

Quantification of circulating TCR-engineered T cells targeting a human endogenous retrovirus post-adoptive transfer using nanoplate digital PCR.

expansion of genetically modified T cells in cancer patients following adoptive transfer has been linked to both anti-tumor activity and T cell-mediated toxicities. The development of digital PCR has improved the accuracy in quantifying the status of adoptively infused T cells compared to qPCR or flow cytometry. Here, we developed and evaluated the feasibility and performance of nanoplate-based digital PCR (ndPCR) to quantify adoptively infused T cells engineered with a T cell receptor (TCR) that recognizes a human endogenous retrovirus type E (HERV-E) antigen.

Regression of renal cell carcinoma by T cell receptor-engineered T cells targeting a human endogenous retrovirus.

Background: We discovered a novel human endogenous retrovirus (CT-RCC HERV-E) that was selectively expressed in most clear cell renal cell carcinomas (ccRCC) and served as a source of antigens for T cell-mediated killing. Here, we described the cloning of a novel T cell receptor (TCR) targeting a CT-RCC HERV-E-derived antigen specific to ccRCC and characterized antitumor activity of HERV-E TCR-transduced T cells (HERV-E T cells).

Methods: We isolated a CD8 T cell clone from a patient with immune-mediated regression of ccRCC post-allogeneic stem cell transplant that recognized the CT-RCC-1 HERV-E-derived peptide in an HLA-A11-restricted manner.

Social Determinants modulate NK cell activity via obesity, LDL, and DUSP1 signaling.

Adverse social determinants of health (aSDoH) are associated with obesity and related comorbidities like diabetes, cardiovascular disease, and cancer. Obesity is also associated with natural killer cell (NK) dysregulation, suggesting a potential mechanistic link. Therefore, we measured NK phenotypes and function in a cohort of African-American (AA) women from resource-limited neighborhoods.

Spleen tyrosine kinase inhibition restores myeloid homeostasis in COVID-19.

Spleen tyrosine kinase (SYK) is a previously unidentified therapeutic target that inhibits neutrophil and macrophage activation in coronavirus disease 2019 (COVID-19). Fostamatinib, a SYK inhibitor, was studied in a phase 2 placebo-controlled randomized clinical trial and was associated with improvements in many secondary end points related to efficacy. Here, we used a multiomic approach to evaluate cellular and soluble immune mediator responses of patients enrolled in this trial.

LIR-1 educates expanded human NK cells and defines a unique antitumor NK cell subset with potent antibody-dependent cellular cytotoxicity.

Objective: KIR and NKG2A receptors educate human NK cells to stay responsive to cells with diminished HLA class I. Here, we addressed whether the HLA class I-binding receptor LIR-1 (LILRB1/ILT2/CD85j), which is widely expressed on human NK cells, can mediate education and contribute to antitumor functions of NK cells.

Methods: Healthy donor NK cells either unstimulated, overnight cytokine-activated or -expanded were used to target human cell lines.

Pre-vaccination and early B cell signatures predict antibody response to SARS-CoV-2 mRNA vaccine.

SARS-CoV-2 mRNA vaccines are highly effective, although weak antibody responses are seen in some individuals with correlates of immunity that remain poorly understood. Here we longitudinally dissected antibody, plasmablast, and memory B cell (MBC) responses to the two-dose Moderna mRNA vaccine in SARS-CoV-2-uninfected adults. Robust, coordinated IgA and IgG antibody responses were preceded by bursts of spike-specific plasmablasts after both doses, but earlier and more intensely after dose two.

Combined haploidentical and cord blood transplantation for refractory severe aplastic anaemia and hypoplastic myelodysplastic syndrome.

Umbilical cord blood (UCB) transplantation is a potentially curative treatment for patients with refractory severe aplastic anaemia (SAA), but has historically been associated with delayed engraftment and high graft failure and mortality rates. We conducted a prospective phase 2 trial to assess outcome of an allogeneic transplant regimen that co-infused a single UCB unit with CD34 -selected cells from a haploidentical relative. Among 29 SAA patients [including 10 evolved to myelodysplastic syndrome (MDS)] who underwent the haplo cord transplantation (median age 20 years), 97% had neutrophil recovery (median 10 days), and 93% had platelet recovery (median 32 days).

RNA-Seq Analysis Reveals CCR5 as a Key Target for CRISPR Gene Editing to Regulate In Vivo NK Cell Trafficking.

A growing number of natural killer (NK) cell-based immunotherapy trials utilize ex vivo expansion to grow and activate allogenic and autologous NK cells prior to administration to patients with malignancies. Recent data in both murine and macaque models have shown that adoptively infused ex vivo expanded NK cells have extensive trafficking into liver tissue, with relatively low levels of homing to other sites where tumors often reside, such as the bone marrow or lymph nodes. Here, we evaluated gene and surface expression of molecules involved in cellular chemotaxis in freshly isolated human NK cells compared with NK cells expanded ex vivo using two different feeder cells lines: Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCLs) or K562 cells with membrane-bound (mb) 4-1BB ligand and interleukin (IL)-21.

Systematic improvements in lentiviral transduction of primary human natural killer cells undergoing expansion.

Transduction of primary human natural killer (NK) cells with lentiviral vectors has historically been challenging. We sought to evaluate multiple parameters to optimize lentiviral transduction of human peripheral blood NK cells being expanded to large numbers using a good manufacturing practice (GMP)-compliant protocol that utilizes irradiated lymphoblastoid (LCL) feeder cells. Although prestimulation of NK cells with interleukin (IL)-2 for 2 or more days facilitated transduction with vesicular stomatitis virus glycoprotein (VSVG)-pseudotyped lentivirus, there was a subsequent impairment in the capacity of transduced NK cells to proliferate when stimulated with LCL feeder cells.

Intrabone transplantation of CD34+ cells with optimized delivery does not enhance engraftment in a rhesus macaque model.

Intrabone (IB) injection of umbilical cord blood has been proposed as a potential mechanism to improve transplant engraftment and prevent graft failure. However, conventional IB techniques produce low retention of transplanted cells in the marrow. To overcome this barrier, we developed an optimized IB (OIB) injection method using low-volume, computer-controlled slow infusion that promotes cellular retention in the marrow.

Genome-Wide Analysis of Off-Target CRISPR/Cas9 Activity in Single-Cell-Derived Human Hematopoietic Stem and Progenitor Cell Clones.

CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9)-mediated genome editing holds remarkable promise for the treatment of human genetic diseases. However, the possibility of off-target Cas9 activity remains a concern. To address this issue using clinically relevant target cells, we electroporated Cas9 ribonucleoprotein (RNP) complexes (independently targeted to two different genomic loci, the locus on chromosome 2 and the locus on chromosome 19) into human mobilized peripheral blood-derived hematopoietic stem and progenitor cells (HSPCs) and assessed the acquisition of somatic mutations in an unbiased, genome-wide manner via whole genome sequencing (WGS) of single-cell-derived HSPC clones.

Augmentation of NK Cell Proliferation and Anti-tumor Immunity by Transgenic Expression of Receptors for EPO or TPO.

Many investigational adoptive immunotherapy regimens utilizing natural killer (NK) cells require the administration of interleukin-2 (IL-2) or IL-15, but these cytokines cause serious dose-dependent toxicities. To reduce or preclude the necessity for IL-2 use, we investigated whether genetic engineering of NK cells to express the erythropoietin (EPO) receptor (EPOR) or thrombopoietin (TPO) receptor (c-MPL) could be used as a method to improve NK cell survival and function. Viral transduction of NK-92 cells to express EPOR or c-MPL receptors conveyed signaling via appropriate pathways, protected cells from apoptosis, augmented cellular proliferation, and increased cell cytotoxic function in response to EPO or TPO ligands in vitro.

A pilot trial of complement inhibition using eculizumab to overcome platelet transfusion refractoriness in human leukocyte antigen allo-immunized patients.

Heavily transfused patients frequently develop human leukocyte antigen (HLA) allo-immunization resulting in platelet transfusion refractoriness and a high risk for life-threatening thrombocytopenia. Data suggest complement activation leading to the destruction of platelets bound by HLA allo-antibodies may play a pathophysiologic role in platelet refractoriness. Here we conducted a pilot trial to investigate the use of eculizumab, a monoclonal antibody that binds and inhibits C5 complement, to treat platelet transfusion refractoriness in allo-immunized patients with severe thrombocytopenia.

Tracking of Adoptively Transferred Natural Killer Cells in Rhesus Macaques Using Zirconium-Oxine Cell Labeling and PET Imaging.

Purpose: Trials of adoptive natural killer (NK)-cell immunotherapy for hematologic malignancies have thus far shown only marginal effects, despite the potent antitumor activity of these cells. Homing of infused cells to tumor microenvironments is critical for efficacy, but has not been well characterized. We established a novel method to track and quantify the distribution of adoptively transferred NK cells using rhesus macaques (RM) as a clinically relevant preclinical model.

Enhanced Bone Marrow Homing of Natural Killer Cells Following mRNA Transfection With Gain-of-Function Variant CXCR4.

Adoptive transfer of natural killer (NK) cells can induce remission in patients with relapsed/refractory leukemia and myeloma. However, to date, clinical efficacy of NK cell immunotherapy has been limited to a sub-fraction of patients. Here we show that steps incorporated in the manipulation/production of NK cell products used for adoptive infusion, such as over-night IL-2 activation or cryopreservation followed by expansion, drastically decreases NK cell surface expression of the bone marrow (BM) homing chemokine receptor CXCR4.

Bortezomib sensitizes multiple myeloma to NK cells via ER-stress-induced suppression of HLA-E and upregulation of DR5.

Although the proteasome inhibitor bortezomib has significantly improved the survival of patients with multiple myeloma (MM), the disease remains fatal as most patients eventually develop progressive disease. Recent data indicate that MM cells can evade bortezomib-induced cell death by undergoing autophagy as a consequence of endoplasmatic reticulum (ER)-stress induced by proteasome inhibition. Here we show that bortezomib sensitizes MM cells to NK cell killing via two distinct mechanisms: a) upregulation of the TRAIL death receptor DR5 on the surface of MM cells and b) ER-stress induced reduction of cell surface HLA-E.

Allogeneic transplantation using CD34 selected peripheral blood progenitor cells combined with non-mobilized donor T cells for refractory severe aplastic anaemia.

Allogeneic haematopoietic stem cell transplantation is curative for severe aplastic anaemia (SAA) unresponsive to immunosuppressive therapy. To reduce chronic graft-versus-host disease (GVHD), which occurs more frequently after peripheral blood stem cell (PBSC) transplantation compared to bone-marrow transplantation (BMT), and to prevent graft rejection, we developed a novel partial T-cell depleted transplant that infuses high numbers of granulocyte colony-stimulating factor-mobilized CD34 selected PBSCs combined with a BMT-equivalent dose of non-mobilized donor T-cells. Fifteen patients with refractory SAA received cyclophosphamide, anti-thymocyte globulin and fludarabine conditioning, and were transplanted with a median 8 × 10 CD34  cells/kg and 2 × 10 non-mobilized CD3 T-cells/kg from human leucocyte antigen-matched sibling donors.

Effect of high-dose plerixafor on CD34 cell mobilization in healthy stem cell donors: results of a randomized crossover trial.

Hematopoietic stem cells can be mobilized from healthy donors using single-agent plerixafor without granulocyte colony-stimulating factor and, following allogeneic transplantation, can result in sustained donor-derived hematopoiesis. However, when a single dose of plerixafor is administered at a conventional 240 μg/kg dose, approximately one-third of donors will fail to mobilize the minimally acceptable dose of CD34 cells needed for allogeneic transplantation. We conducted an open-label, randomized trial to assess the safety and activity of high-dose (480 μg/kg) plerixafor in CD34 cell mobilization in healthy donors.

Checkpoint Inhibition of KIR2D with the Monoclonal Antibody IPH2101 Induces Contraction and Hyporesponsiveness of NK Cells in Patients with Myeloma.

Purpose: Immune checkpoint inhibitors have recently revolutionized cancer immunotherapy. On the basis of data showing KIR-ligand mismatched natural killer (NK) cells reduce the risk of leukemia and multiple myeloma relapse following allogeneic hematopoietic stem cell transplantation, investigators have developed a checkpoint inhibition antibody that blocks KIR on NK cells. Although in vitro studies suggest the KIR2D-specific antibody IPH2101 induces KIR-ligand mismatched tumor killing by NK cells, our single-arm phase II clinical trial in patients with smoldering multiple myeloma was prematurely terminated due to lack of clinical efficacy.

Efficient mRNA-Based Genetic Engineering of Human NK Cells with High-Affinity CD16 and CCR7 Augments Rituximab-Induced ADCC against Lymphoma and Targets NK Cell Migration toward the Lymph Node-Associated Chemokine CCL19.

For more than a decade, investigators have pursued methods to genetically engineer natural killer (NK) cells for use in clinical therapy against cancer. Despite considerable advances in viral transduction of hematopoietic stem cells and T cells, transduction efficiencies for NK cells have remained disappointingly low. Here, we show that NK cells can be genetically reprogramed efficiently using a cGMP-compliant mRNA electroporation method that induces rapid and reproducible transgene expression in nearly all transfected cells, without negatively influencing their viability, phenotype, and cytotoxic function.

Documenting stress in caregivers of transplantation patients: initial evidence of HPA dysregulation.

There is growing evidence linking caregiver stress with an increased risk for morbidity and mortality. While the emotional and practical burden experienced by caregivers is well established, the physiological changes that may affect the caregiver's health are less understood. This study sought to compare self-reported stress, anxiety and depression along with neuroendocrine and immune markers of stress among adult caregivers of allogeneic hematopoietic stem cell transplantation patients during the acute transplant recovery period to matched non-caregivers controls.