Interleukin-21 engineering enhances NK cell activity against glioblastoma via CEBPD.

Glioblastoma (GBM) is an aggressive brain cancer with limited therapeutic options. Natural killer (NK) cells are innate immune cells with strong anti-tumor activity and may offer a promising treatment strategy for GBM. We compared the anti-GBM activity of NK cells engineered to express interleukin (IL)-15 or IL-21. Using multiple in vivo models, IL-21 NK cells were superior to IL-15 NK cells both in terms of safety and long-term anti-tumor activity, with locoregionally administered IL-15 NK cells proving toxic and ineffective at tumor control. IL-21 NK cells displayed a unique chromatin accessibility signature, with CCAAT/enhancer-binding proteins (C/EBP), especially CEBPD, serving as key transcription factors regulating their enhanced function. Deletion of CEBPD resulted in loss of IL-21 NK cell potency while its overexpression increased NK cell long-term cytotoxicity and metabolic fitness. These results suggest that IL-21, through C/EBP transcription factors, drives epigenetic reprogramming of NK cells, enhancing their anti-tumor efficacy against GBM.

CD28 costimulation augments CAR signaling in NK cells via the LCK/CD3Z/ZAP70 signaling axis.

Multiple factors in the design of a chimeric antigen receptor (CAR) influence CAR T-cell activity, with costimulatory signals being a key component. Yet, the impact of costimulatory domains on the downstream signaling and subsequent functionality of CAR-engineered natural killer (NK) cells remains largely unexplored. Here, we evaluated the impact of various costimulatory domains on CAR-NK cell activity, using a CD70-targeting CAR. We found that CD28, a costimulatory molecule not inherently present in mature NK cells, significantly enhanced the antitumor efficacy and long-term cytotoxicity of CAR-NK cells both in vitro and in multiple xenograft models of hematologic and solid tumors. Mechanistically, we showed that CD28 linked to CD3Z creates a platform that recruits critical kinases, such as LCK and ZAP70, initiating a signaling cascade that enhances CAR-NK cell function. Our study provides insights into how CD28 costimulation enhances CAR-NK cell function and supports its incorporation in NK-based CARs for cancer immunotherapy.

Activated B cells suppress T-cell function through metabolic competition.

BACKGROUND: B cells play a pivotal role in regulating the immune response. The induction of B cell-mediated immunosuppressive function requires B cell activating signals. However, the mechanisms by which activated B cells mediate T-cell suppression are not fully understood. METHODS: We investigated the potential contribution of metabolic activity of activated B cells to T-cell suppression by performing in vitro experiments and by analyzing clinical samples using mass cytometry and single-cell RNA sequencing. RESULTS: Here we show that following activation, B cells acquire an immunoregulatory phenotype and promote T-cell suppression by metabolic competition. Activated B cells induced hypoxia in T cells in a cell-cell contact dependent manner by consuming more oxygen via an increase in their oxidative phosphorylation (OXPHOS). Moreover, activated B cells deprived T cells of glucose and produced lactic acid through their high glycolytic activity. Activated B cells thus inhibited the mammalian target of rapamycin pathway in T cells, resulting in suppression of T-cell cytokine production and proliferation. Finally, we confirmed the presence of tumor-associated B cells with high glycolytic and OXPHOS activities in patients with melanoma, associated with poor response to immune checkpoint blockade therapy. CONCLUSIONS: We have revealed for the first time the immunomodulatory effects of the metabolic activity of activated B cells and their possible role in suppressing antitumor T-cell responses. These findings add novel insights into immunometabolism and have important implications for cancer immunotherapy.

Generation of glucocorticoid-resistant SARS-CoV-2 T cells for adoptive cell therapy.

Adoptive cell therapy with virus-specific T cells has been used successfully to treat life-threatening viral infections, supporting application of this approach to coronavirus disease 2019 (COVID-19). We expand severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) T cells from the peripheral blood of COVID-19-recovered donors and non-exposed controls using different culture conditions. We observe that the choice of cytokines modulates the expansion, phenotype, and hierarchy of antigenic recognition by SARS-CoV-2 T cells. Culture with interleukin (IL)-2/4/7, but not under other cytokine-driven conditions, results in more than 1,000-fold expansion in SARS-CoV-2 T cells with a retained phenotype, function, and hierarchy of antigenic recognition compared with baseline (pre-expansion) samples. Expanded cytotoxic T lymphocytes (CTLs) are directed against structural SARS-CoV-2 proteins, including the receptor-binding domain of Spike. SARS-CoV-2 T cells cannot be expanded efficiently from the peripheral blood of non-exposed controls. Because corticosteroids are used for management of severe COVID-19, we propose an efficient strategy to inactivate the glucocorticoid receptor gene (NR3C1) in SARS-CoV-2 CTLs using CRISPR-Cas9 gene editing.

Targeting the αv integrin/TGF-ß axis improves natural killer cell function against glioblastoma stem cells.

Glioblastoma multiforme (GBM), the most aggressive brain cancer, recurs because glioblastoma stem cells (GSCs) are resistant to all standard therapies. We showed that GSCs, but not normal astrocytes, are sensitive to lysis by healthy allogeneic natural killer (NK) cells in vitro. Mass cytometry and single-cell RNA sequencing of primary tumor samples revealed that GBM tumor-infiltrating NK cells acquired an altered phenotype associated with impaired lytic function relative to matched peripheral blood NK cells from patients with GBM or healthy donors. We attributed this immune evasion tactic to direct cell-to-cell contact between GSCs and NK cells via αv integrin-mediated TGF-ß activation. Treatment of GSC-engrafted mice with allogeneic NK cells in combination with inhibitors of integrin or TGF-ß signaling or with TGFBR2 gene-edited allogeneic NK cells prevented GSC-induced NK cell dysfunction and tumor growth. These findings reveal an important mechanism of NK cell immune evasion by GSCs and suggest the αv integrin/TGF-ß axis as a potentially useful therapeutic target in GBM.

Metabolic Reprogramming of GMP Grade Cord Tissue Derived Mesenchymal Stem Cells Enhances Their Suppressive Potential in GVHD.

Acute graft-vs.-host (GVHD) disease remains a common complication of allogeneic stem cell transplantation with very poor outcomes once the disease becomes steroid refractory. Mesenchymal stem cells (MSCs) represent a promising therapeutic approach for the treatment of GVHD, but so far this strategy has had equivocal clinical efficacy. Therapies using MSCs require optimization taking advantage of the plasticity of these cells in response to different microenvironments. In this study, we aimed to optimize cord blood tissue derived MSCs (CBti MSCs) by priming them using a regimen of inflammatory cytokines. This approach led to their metabolic reprogramming with enhancement of their glycolytic capacity. Metabolically reprogrammed CBti MSCs displayed a boosted immunosuppressive potential, with superior immunomodulatory and homing properties, even after cryopreservation and thawing. Mechanistically, primed CBti MSCs significantly interfered with glycolytic switching and mTOR signaling in T cells, suppressing T cell proliferation and ensuing polarizing toward T regulatory cells. Based on these data, we generated a Good Manufacturing Process (GMP) Laboratory protocol for the production and cryopreservation of primed CBti MSCs for clinical use. Following thawing, these cryopreserved GMP-compliant primed CBti MSCs significantly improved outcomes in a xenogenic mouse model of GVHD. Our data support the concept that metabolic profiling of MSCs can be used as a surrogate for their suppressive potential in conjunction with conventional functional methods to support their therapeutic use in GVHD or other autoimmune disorders.

Combining AFM13, a Bispecific CD30/CD16 Antibody, with Cytokine-Activated Blood and Cord Blood-Derived NK Cells Facilitates CAR-like Responses Against CD30+ Malignancies.

PURPOSE: Natural killer (NK)-cell recognition and function against NK-resistant cancers remain substantial barriers to the broad application of NK-cell immunotherapy. Potential solutions include bispecific engagers that target NK-cell activity via an NK-activating receptor when simultaneously targeting a tumor-specific antigen, as well as enhancing functionality using IL12/15/18 cytokine pre-activation. EXPERIMENTAL DESIGN: We assessed single-cell NK-cell responses stimulated by the tetravalent bispecific antibody AFM13 that binds CD30 on leukemia/lymphoma targets and CD16A on various types of NK cells using mass cytometry and cytotoxicity assays. The combination of AFM13 and IL12/15/18 pre-activation of blood and cord blood-derived NK cells was investigated in vitro and in vivo. RESULTS: We found heterogeneity within AFM13-directed conventional blood NK cell (cNK) responses, as well as consistent AFM13-directed polyfunctional activation of mature NK cells across donors. NK-cell source also impacted the AFM13 response, with cNK cells from healthy donors exhibiting superior responses to those from patients with Hodgkin lymphoma. IL12/15/18-induced memory-like NK cells from peripheral blood exhibited enhanced killing of CD30+ lymphoma targets directed by AFM13, compared with cNK cells. Cord-blood NK cells preactivated with IL12/15/18 and ex vivo expanded with K562-based feeders also exhibited enhanced killing with AFM13 stimulation via upregulation of signaling pathways related to NK-cell effector function. AFM13-NK complex cells exhibited enhanced responses to CD30+ lymphomas in vitro and in vivo. CONCLUSIONS: We identify AFM13 as a promising combination with cytokine-activated adult blood or cord-blood NK cells to treat CD30+ hematologic malignancies, warranting clinical trials with these novel combinations.

GMP-Compliant Universal Antigen Presenting Cells (uAPC) Promote the Metabolic Fitness and Antitumor Activity of Armored Cord Blood CAR-NK Cells.

Natural killer (NK) cells are innate lymphocytes recognized for their important role against tumor cells. NK cells expressing chimeric antigen receptors (CARs) have enhanced effector function against various type of cancer and are attractive contenders for the next generation of cancer immunotherapies. However, a number of factors have hindered the application of NK cells for cellular therapy, including their poor in vitro growth kinetics and relatively low starting percentages within the mononuclear cell fraction of peripheral blood or cord blood (CB). To overcome these limitations, we genetically-engineered human leukocyte antigen (HLA)-A- and HLA-B- K562 cells to enforce the expression of CD48, 4-1BBL, and membrane-bound IL-21 (mbIL21), creating a universal antigen presenting cell (uAPC) capable of stimulating their cognate receptors on NK cells. We have shown that uAPC can drive the expansion of both non-transduced (NT) and CAR-transduced CB derived NK cells by >900-fold in 2 weeks of co-culture with excellent purity (>99.9%) and without indications of senescence/exhaustion. We confirmed that uAPC-expanded research- and clinical-grade NT and CAR-transduced NK cells have higher metabolic fitness and display enhanced effector function against tumor targets compared to the corresponding cell fractions cultured without uAPCs. This novel approach allowed the expansion of highly pure GMP-grade CAR NK cells at optimal cell numbers to be used for adoptive CAR NK cell-based cancer immunotherapy.

Targeting a cytokine checkpoint enhances the fitness of armored cord blood CAR-NK cells.

Immune checkpoint therapy has resulted in remarkable improvements in the outcome for certain cancers. To broaden the clinical impact of checkpoint targeting, we devised a strategy that couples targeting of the cytokine-inducible Src homology 2-containing (CIS) protein, a key negative regulator of interleukin 15 (IL-15) signaling, with fourth-generation "armored" chimeric antigen receptor (CAR) engineering of cord blood-derived natural killer (NK) cells. This combined strategy boosted NK cell effector function through enhancing the Akt/mTORC1 axis and c-MYC signaling, resulting in increased aerobic glycolysis. When tested in a lymphoma mouse model, this combined approach improved NK cell antitumor activity more than either alteration alone, eradicating lymphoma xenografts without signs of any measurable toxicity. We conclude that targeting a cytokine checkpoint further enhances the antitumor activity of IL-15-secreting armored CAR-NK cells by promoting their metabolic fitness and antitumor activity. This combined approach represents a promising milestone in the development of the next generation of NK cells for cancer immunotherapy.

Large-scale GMP-compliant CRISPR-Cas9-mediated deletion of the glucocorticoid receptor in multivirus-specific T cells.

Virus-specific T cells have proven highly effective for the treatment of severe and drug-refractory infections after hematopoietic stem cell transplant (HSCT). However, the efficacy of these cells is hindered by the use of glucocorticoids, often given to patients for the management of complications such as graft-versus-host disease. To address this limitation, we have developed a novel strategy for the rapid generation of good manufacturing practice (GMP)-grade glucocorticoid-resistant multivirus-specific T cells (VSTs) using clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) gene-editing technology. We have shown that deleting the nuclear receptor subfamily 3 group C member 1 (NR3C1; the gene encoding for the glucocorticoid receptor) renders VSTs resistant to the lymphocytotoxic effect of glucocorticoids. NR3C1-knockout (KO) VSTs kill their targets and proliferate successfully in the presence of high doses of dexamethasone both in vitro and in vivo. Moreover, we developed a protocol for the rapid generation of GMP-grade NR3C1 KO VSTs with high on-target activity and minimal off-target editing. These genetically engineered VSTs promise to be a novel approach for the treatment of patients with life-threatening viral infections post-HSCT on glucocorticoid therapy.

Use of CAR-Transduced Natural Killer Cells in CD19-Positive Lymphoid Tumors.

BACKGROUND: Anti-CD19 chimeric antigen receptor (CAR) T-cell therapy has shown remarkable clinical efficacy in B-cell cancers. However, CAR T cells can induce substantial toxic effects, and the manufacture of the cells is complex. Natural killer (NK) cells that have been modified to express an anti-CD19 CAR have the potential to overcome these limitations. METHODS: In this phase 1 and 2 trial, we administered HLA-mismatched anti-CD19 CAR-NK cells derived from cord blood to 11 patients with relapsed or refractory CD19-positive cancers (non-Hodgkin's lymphoma or chronic lymphocytic leukemia [CLL]). NK cells were transduced with a retroviral vector expressing genes that encode anti-CD19 CAR, interleukin-15, and inducible caspase 9 as a safety switch. The cells were expanded ex vivo and administered in a single infusion at one of three doses (1×105, 1×106, or 1×107 CAR-NK cells per kilogram of body weight) after lymphodepleting chemotherapy. RESULTS: The administration of CAR-NK cells was not associated with the development of cytokine release syndrome, neurotoxicity, or graft-versus-host disease, and there was no increase in the levels of inflammatory cytokines, including interleukin-6, over baseline. The maximum tolerated dose was not reached. Of the 11 patients who were treated, 8 (73%) had a response; of these patients, 7 (4 with lymphoma and 3 with CLL) had a complete remission, and 1 had remission of the Richter's transformation component but had persistent CLL. Responses were rapid and seen within 30 days after infusion at all dose levels. The infused CAR-NK cells expanded and persisted at low levels for at least 12 months. CONCLUSIONS: Among 11 patients with relapsed or refractory CD19-positive cancers, a majority had a response to treatment with CAR-NK cells without the development of major toxic effects. (Funded by the M.D. Anderson Cancer Center CLL and Lymphoma Moonshot and the National Institutes of Health; ClinicalTrials.gov number, NCT03056339.).

Primary Thrombophilia in México XI: Activated Protein C Resistance Phenotypes are Multifactorial.

Activated protein C resistance (aPCR) phenotypes represent around 20% of the laboratory findings in Mexican Mestizos having suffered thrombosis and displaying clinical markers of thrombophilia. In a single institution for a 276-month period, 96 Mexican mestizos with a history of thrombosis and clinical markers of a primary thrombophilic state were prospectively studied to identify a thrombophilic condition. An abnormal aPCR phenotype was identified in 18 individuals. Evaluation of those with an abnormal aPCR phenotype, identified that 44% had factor V Leiden mutation, 22% increased levels of factor VIII, 16% anti-phospholipid antibodies and 6% a lupus anticoagulant. In the remaining 22%, the use of direct oral anticoagulants (DOACs) in the past period of 12-24 h was recorded. We found significant associations between abnormal aPCR phenotype and the factor V Leiden mutation (p = 0022), between abnormal aPCR phenotype and the use of DOACs (p = 0.006) and between antiphospholipid antibodies and lupus anticoagulant (p < 0.0001). These data are consonant with those observed in other populations and further identify that consideration be given to identifying whether individuals are being treated with the novel DOACs when conducting laboratory studies oriented to identify the etiology of thrombosis.

A Feasibility Study of the Full Outpatient Conduction of Hematopoietic Transplants in Persons with Multiple Sclerosis Employing Autologous Non-Cryopreserved Peripheral Blood Stem Cells.

BACKGROUND: With the goal of achieving immune system reset, autologous hematopoietic stem cell transplantations have been performed in patients with multiple sclerosis (MS). MATERIAL AND METHODS: Two hundred and eighty-six consecutive patients with MS were autografted in a single center using non-frozen peripheral blood stem cells (PBSCs), on an outpatient basis and conditioning with cyclophosphamide and rituximab. The protocol was registered in ClinicalTrials.gov identifier NCT02674217. RESULTS: One hundred and ninety-four females and 92 males were included; the median age was 47. All procedures were started on an outpatient basis and only 8 persons needed to be admitted to the hospital during the procedure. In order to obtain at least 1 × 106/kg viable CD34 cells, 1-4 aphereses were performed (median 1). The total number of viable CD34+ cells infused ranged between 1 and 19.2 × 106/kg (median 4.6). Patients recovered above 0.5 × 109/L absolute granulocytes on median day 8 (range 0-12). Two individuals needed red blood cells but none needed platelet transfusions. There were no transplant-related deaths and the 128-month overall survival of the patients is 100%. In 82 persons followed up for 3 or more months, the Expanded Disability Status Scale diminished from a mean of 5.2-4.9, the best results being obtained in relapsing-remitting and primary progressive MS. CONCLUSIONS: It is possible to conduct autotransplants for patients with MS employing non-frozen PBSCs and outpatient conduction. Additional information is needed to assess the efficacy of these procedures in the treatment of patients with MS.

Primary Thrombophilia in Mexico XII: Miscarriages Are More Frequent in People with Sticky Platelet Syndrome.

OBJECTIVE: Sticky platelet syndrome (SPS) is an inherited condition that leads to arterial and venous thrombosis. There is scant information about the association between SPS and obstetric complications. This study aimed to assess the relationship between SPS and fetal loss at a single institution. MATERIALS AND METHODS: The obstetric histories of all consecutive female patients prospectively studied in a 324-month period at a single institution with a history of thrombosis and a clinical marker of primary thrombophilia were reviewed. RESULTS: Between 1989 and 2016, 268 consecutive patients with a clinical marker of primary thrombophilia and a history of arterial or venous thrombosis were studied; of these, 108 were female patients. Within this subset of thrombophilic females, 77 (71%) had been pregnant at some point. Twenty-eight of these 77 patients (37%) had had a spontaneous abortion and 24 of those (86%) were found to have SPS. On the other hand, in a subset of 73 female patients with SPS who had been pregnant, 32% had miscarriages. These figures are significantly higher than the prevalence of spontaneous abortions in the general Mexican population of pregnant women, which is 12%-13% (chi-square: 7.47; p=0.0063). Accordingly, the relative risk of having a miscarriage is 2.66 times higher in female patients with SPS than in the general population (p=0.0014). CONCLUSION: In Mexico, female patients with SPS experience significantly more spontaneous abortions than the general population. Since the treatment of SPS is simple and effective and could in turn prevent adverse obstetric outcomes, its investigation in women treated for obstetric complications may be useful and deserves further research.

More on the thrombocytopenia of the non-alcoholic fatty liver disease.

BACKGROUND: Using only serologic determinations, we have previously found that thrombocytopenia presents in less than one half of patients with non-alcoholic fatty liver disease (NAFLD). MATERIAL AND METHODS: Employing a more accurate method to define the presence of NAFLD, serologic determinations (Fibromax®) coupled with liver transient elastography (TE/Fibroscan®), we have prospectively studied a group of 211 individuals with a suspicion of a liver disease. RESULTS: NAFLD was identified in 81 individuals. In 48 persons another causes of liver damage were identified and discarded from further analysis. A subset of 33 patients with NAFLD without liver fibrosis or cirrhosis was analyzed. In eight of them (24%), thrombocytopenia (less than 150 × 109/l platelets) was identified. The presence of thrombocytopenia in this subset of persons was associated with overweight, was usually mild, above 50 × 109/l, was not associated to mucocutaneous bleeding and did not require treatment. CONCLUSIONS: NAFLD should be considered as a cause of mild thrombocytopenia. Our initial observation has been confirmed and defined more precisely. Additional studies are needed to further define more features of the thrombocytopenia of NAFLD, as well as its mechanisms.

Mexican Biosimilar Filgrastim for Autologous Hematopoietic Stem Cell Mobilization and Transplantation.

BACKGROUND: Following the release of the initial presentation of filgrastim (granulocyte colony-stimulating factor), several biosimilars have been developed worldwide. OBJECTIVE: To study the efficacy of a Mexican biosimilar granulocyte colony-stimulating factor in a single transplant center. METHODS: In a group of 19 consecutive patients with multiple sclerosis given autografts, we employed granulocyte colony-stimulating factors to mobilize stem cells from the bone marrow to the peripheral blood, either the original granulocyte colony-stimulating factor (n = 10) or a Mexican granulocyte colony-stimulating factor biosimilar (n = 9). RESULTS: The efficacy of both agents was similar in mobilization capacity, white blood cell count rise, stem cell collection, and kinetics of auto-engraftment. CONCLUSION: We conclude that both granulocyte colony-stimulating factor agents were similar in their efficacy to mobilize stem cells and usefulness in autografts.

IKAROS Gene Deleted B-Cell Acute Lymphoblastic Leukemia in Mexican Mestizos: Observations in Seven Patients and a Short Review of the Literature.

BACKGROUND: In B-cell acute lymphoblastic leukemia, one of the most frequent cytogenetic alterations is the presence of the Philadelphia chromosome. Recently, newly identified genetic alterations have been studied, among them the IKZF1 deletion. IKZF1 encodes IKAROS, a zinc finger protein that plays an important role in hematopoiesis involving the regulation process of adhesion, cellular migration, and as a tumor suppressor. OBJECTIVE: We aimed to study the impact of IKAROS deletion in the evolution and prognosis of B-cell acute lymphoblastic leukemia. MATERIALS AND METHODS: At a single center we prospectively studied patients diagnosed with B-cell acute lymphoblastic leukemia and screened for IKZF1 deletion using the multiplex ligation-dependent probe amplification method. We did a descriptive analysis of patients positive for the IKZF1 deletion to determine its impact on the evolution of the disease and survival rate. RESULTS: Between 2010 and 2015, 16 Mexican mestizo patients with B-cell acute lymphoblastic leukemia were prospectively screened for IKZF1 deletion; seven (43%) were positive and were included for further analysis. The age range of patients was 13-60 years; six were males and one female. All cases had type B acute lymphoblastic leukemia. Of the seven patients, two died, three were lost to follow-up, and two continue in complete remission with treatment. Results are worse than those in a group of patients with non-mutated IKAROS B-cell acute lymphoblastic leukemia previously studied in our center. CONCLUSIONS: Although this is a small sample, the presence of IKAROS deletion in acute lymphoblastic leukemia patients could represent a poor-prognosis marker and was probably related to therapy failure. It is also possible that this variant of leukemia may be more prevalent in Mexico. More studies are needed to define the role of IKZF1 deletion in acute lymphoblastic leukemia and the real prevalence of the disease in different populations.