NKG2A genetic deletion promotes human primary NK cell anti-tumor responses better than an anti-NKG2A monoclonal antibody.

Natural killer (NK) cells eliminate infected or cancer cells via their cytotoxic capacity. NKG2A is an inhibitory receptor on NK cells and cancer cells often overexpress its ligand HLA-E to evade NK cell surveillance. Given the successes of immune checkpoint blockade in cancer therapy, NKG2A is an interesting novel target. However, anti-NKG2A antibodies have shown limited clinical response. In the pursuit of enhancing NK cell-mediated anti-tumor responses, we devised a Cas9-based strategy to delete KLRC1, encoding NKG2A, in human primary NK cells. Our approach involved electroporation of KLRC1-targeting Cas9 ribonucleoprotein resulting in effective ablation of NKG2A expression. Compared with anti-NKG2A antibody blockade, NKG2AKO NK cells exhibited enhanced activation, reduced suppressive signaling, and elevated expression of key transcription factors. NKG2AKO NK cells overcame inhibition from HLA-E, significantly boosting NK cell activity against solid and hematologic cancer cells. We validated this efficacy across multiple cell lines, a xenograft mouse model, and primary human leukemic cells. Combining NKG2A knockout with antibody coating of tumor cells further enhanced cytotoxicity through ADCC. Thus, we provide a comprehensive comparison of inhibition of the NKG2A pathway using genetic ablation and antibodies and provide novel insight in the observed differences in molecular mechanisms, which can be translated to enhance adoptive NK cell immunotherapy.

Low Levels of Serum and Intracellular Vitamin C in Hospitalized COVID-19 Patients.

Vitamin C is a crucial micronutrient for human immune cell function and has potent antioxidant properties. It is hypothesized that vitamin C serum levels decline during infection. However, the precise mechanisms remain unknown. To gain deeper insights into the true role of vitamin C during infections, we aimed to evaluate the body's vitamin C storage during a SARS-CoV-2 infection. In this single-center study, we examined serum and intracellular vitamin C levels in peripheral blood mononuclear cells (PBMCs) of 70 hospitalized COVID-19 patients on the first and fifth days of hospitalization. Also, clinical COVID-19 severity was evaluated at these timepoints. Our findings revealed a high prevalence of hypovitaminosis C and vitamin C deficiency in hospitalized COVID-19 patients (36% and 15%). Moreover, patients with severe or critical disease exhibited a higher prevalence of low serum vitamin C levels than those with moderate illness. Serum vitamin C levels had a weak negative correlation with clinical COVID-19 severity classification on the day of hospitalization; however, there was no correlation with intracellular vitamin C. Intracellular vitamin C levels were decreased in this cohort as compared to a healthy cohort and showed further decline during hospitalization, while serum levels showed no relevant change. Based on this observation, it can be suggested that the reduction of intracellular vitamin C may be attributed to its antioxidative function, the need for replenishing serum levels, or enhanced turnover by immune cells. These data give an incentive to further investigate the role of intracellular vitamin C in a larger and more heterogeneous cohort as well as the underlying mechanisms.

No Effect of Vitamin C Administration on Neutrophil Recovery in Autologous Stem Cell Transplantation for Myeloma or Lymphoma: A Blinded, Randomized Placebo-Controlled Trial.

Vitamin C is an important micronutrient for various immune cells. It increases phagocytic cell function and is necessary for T and natural killer (NK) cell development. Patients in need of an autologous hematopoietic stem cell transplantation (HSCT) are often vitamin C-depleted. We therefore hypothesized that vitamin C supplementation could improve immune recovery in autologous HSCT patients. This blinded, placebo-controlled trial included 44 patients randomized to receive vitamin C or a placebo. The following outcome measures used were clinical and immunological parameters, among others: time to neutrophil recovery, serum, and intracellular vitamin C values. Twenty-one patients received vitamin C, and 23 received a placebo. The time to neutrophil recovery did not differ between the two groups at 11.2 days (p = 0.96). There were no differences in hospitalization time (19.7 vs. 19.1 days, p = 0.80), the incidence of neutropenic fever (57% vs. 78%, p = 0.20), or 3-month overall survival (90.5% vs. 100%, p = 0.13). Bacteremia seemed to occur less in the vitamin C group (10% vs. 35%, p = 0.07). Our study shows no benefit from vitamin C supplementation on neutrophil recovery and hospitalization, despite possible lower rates of bacteremia in the vitamin C group. Therefore, we do not advise vitamin C supplementation in this treatment group.

Easy-to-Use HPLC Method to Measure Intracellular Ascorbic Acid Levels in Human Peripheral Blood Mononuclear Cells and in Plasma.

Given the growing interest in ascorbic acid (AA), there is a need for a reliable and reproducible method to measure AA status in the human body. Serum AA concentrations do not correlate well with tissue levels, but AA levels in leukocytes do. However, a standard method for clinical application is lacking. This present study describes a method to measure AA in the peripheral blood mononuclear cells (PBMCs) with hydrophilic interaction liquid chromatography (HILIC). The method can also be used in plasma and other leukocyte subsets. The measurements of AA in PBMCs and plasma were performed with HPLC with HILIC separation and UV detection. The sample preparation involved the isolation of PBMCs and lysis and precipitation with acetonitrile. European Medicine Agency guidelines for bioanalytic method validation were followed for the evaluation. A highly precise execution of the method was found with intra- and inter-assay variations at a maximum of 7.8%. In 40 healthy donors, a mean intracellular AA concentration of 7.9 microgram/108 cells was found in PBMCs. A correlation between plasma and PBMC AA concentration was not present (r = 0.22). In conclusion, we developed a convenient, reliable, and reproducible method for the quantitative determination of AA within PBMCs and plasma from human blood.

Fractionated Radiotherapy with 3 x 8 Gy Induces Systemic Anti-Tumour Responses and Abscopal Tumour Inhibition without Modulating the Humoral Anti-Tumour Response.

Accumulating evidence indicates that fractionated radiotherapy (RT) can result in distant non-irradiated (abscopal) tumour regression. Although preclinical studies indicate the importance of T cells in this infrequent phenomenon, these studies do not preclude that other immune mechanisms exhibit an addition role in the abscopal effect. We therefore addressed the question whether in addition to T cell mediated responses also humoral anti-tumour responses are modulated after fractionated RT and whether systemic dendritic cell (DC) stimulation can enhance tumour-specific antibody production. We selected the 67NR mammary carcinoma model since this tumour showed spontaneous antibody production in all tumour-bearing mice. Fractionated RT to the primary tumour was associated with a survival benefit and a delayed growth of a non-irradiated (contralateral) secondary tumour. Notably, fractionated RT did not affect anti-tumour antibody titers and the composition of the immunoglobulin (Ig) isotypes. Likewise, we demonstrated that treatment of tumour-bearing Balb/C mice with DC stimulating growth factor Flt3-L did neither modulate the magnitude nor the composition of the humoral immune response. Finally, we evaluated the immune infiltrate and Ig isotype content of the tumour tissue using flow cytometry and found no differences between treatment groups that were indicative for local antibody production. In conclusion, we demonstrate that the 67NR mammary carcinoma in Balb/C mice is associated with a pre-existing antibody response. And, we show that in tumour-bearing Balb/C mice with abscopal tumour regression such pre-existing antibody responses are not altered upon fractionated RT and/or DC stimulation with Flt3-L. Our research indicates that evaluating the humoral immune response in the setting of abscopal tumour regression is not invariably associated with therapeutic effects.

Combination of radiotherapy with the immunocytokine L19-IL2: Additive effect in a NK cell dependent tumour model.

BACKGROUND AND PURPOSE: Recently, we have shown that radiotherapy (RT) combined with the immunocytokine L19-IL2 can induce long-lasting antitumour effects, dependent on ED-B expression and infiltration of cytotoxic T cells. On the other hand, in certain tumours, IL2 treatment can trigger a natural killer cell (NK) immune response. The aim of this study is to investigate the therapeutic effect of our combination therapy in the ED-B positive F9 teratocarcinoma model, lacking MHCI expression and known to be dependent on NK immune responses. MATERIAL AND METHODS: In syngeneic F9 tumour bearing 129/FvHsd mice tumour growth delay was evaluated after local tumour irradiation (10Gy) combined with systemic administration of L19-IL2. Immunological responses were investigated using flow cytometry. RESULTS: Tumour growth delay of L19-IL2 can be further improved by a single dose of RT administered before immunotherapy, but not during immunotherapy. Furthermore, treatment of L19-IL2 favours a NK response and lacks cytotoxic T cell tumour infiltrating immune cells, which may be explained by the absence of MHCI expression. CONCLUSION: An additive effect can be detected when the NK dependent F9 tumour model is treated with radiotherapy and L19-IL2 and therefore this combination could be useful in the absence of tumoural MHCI expression.

Potency of Both Human Th1 and NK Helper Cell Activation is Determined by IL-12p70-Producing PAMP-Matured DCs.

Besides T helper (Th) cells, natural killer (NK) cells have also been described to participate in the shaping of dendritic cell (DC)-mediated adaptive immune responses. At present, it remains unclear to what extent the induction of these NK helper cell immune mechanisms is coupled with Th responses and whether both helper immune responses are induced by the same DC upon specific pathogen recognition receptor (PRR) stimulation. In this study, we demonstrate that maturation of DCs with a cocktail containing FMKp (membrane fragments of Klebsiella pneumoniae) mounts both Th cell and NK cell helper responses in a PRR-triggered dose-dependent manner as determined by the capacity of the helper cells to produce IFN-γ. Furthermore, by triggering an additional PRR pathway [FMKp in combination with poly(I:C) lyovec], we reveal that both approaches modulate the amount of DC-derived IL-12p70 and that this cytokine is the key determinant of the DC-induced Th1 and NK cell helper responses. Moreover, all PRR triggers able to induce IL-12-producing mature DCs are sufficient to induce these helper responses. We propose the existence of a single program used by DCs to induce potent cellular immune responses by stimulating both T helper and NK cell helper processes. This knowledge can help to select the proper PRR triggers in preventive and therapeutic vaccine design.

Prophylactic Interleukin-2 Treatment Prevents Fetal Gut Inflammation and Injury in an Ovine Model of Chorioamnionitis.

BACKGROUND: Chorioamnionitis results from an infection of the fetal membranes and is associated with fetal adverse outcomes notably in the intestine. Using a translational ovine model, we showed that intra-amniotic exposure to inflammatory stimuli decreased the regulatory/effector T (Treg/Teff) cell balance in the gut, which was accompanied by intestinal inflammation and mucosal injury. We thus aimed to augment the Treg/Teff cell ratio in the fetal gut by prophylactic IL-2 treatment and evaluate whether it is sufficient to prevent chorioamnionitis-induced intestinal inflammation and mucosal injury. METHODS: Fetal sheep (122 d of gestation) were intra-amniotically exposed to lipopolysaccharide for 2 or 7 days with or without prophylactic IL-2 treatment (4 d). We evaluated the infiltration of inflammatory cells in the ileum and mesenteric lymph nodes. Cytokine gene expression was analyzed in fetal ileum and the inflammatory changes were correlated with gut wall integrity. RESULTS: IL-2 administration preferentially increased intestinal Treg cells and thus the Treg/Teff cell ratio. Prophylactic IL-2 treatment reduced the lipopolysaccharide-induced influx of neutrophils and CD3(+) T cells and decreased the messenger RNA levels of proinflammatory cytokines including IL-6 and IL-17 in the fetal ileum. Importantly, prophylactic IL-2 treatment prevented mucosal damage without inducing fetal adverse treatment outcomes. CONCLUSIONS: Our data show that prophylactic IL-2 treatment prevents fetal intestinal inflammation and mucosal injury in the context of experimental chorioamnionitis. Modulation of the Treg/Teff cell balance may contribute to the protective effects of IL-2.

Ascorbic acid promotes proliferation of natural killer cell populations in culture systems applicable for natural killer cell therapy.

BACKGROUND AIMS: Natural killer (NK) cell-based immunotherapy is a promising treatment for a variety of malignancies. However, generating sufficient cell numbers for therapy remains a challenge. To achieve this, optimization of protocols is required. METHODS: Mature NK cells were expanded from peripheral blood mononuclear cells PBMCs in the presence of anti-CD3 monoclonal antibody and interleukin-2. Additionally, NK-cell progenitors were generated from CD34(+) hematopoietic stem cells or different T/NK-cell progenitor populations. Generated NK cells were extensively phenotyped, and functionality was determined by means of cytotoxicity assay. RESULTS: Addition of ascorbic acid (AA) resulted in more proliferation of NK cells without influencing NK-cell functionality. In more detail, PBMC-derived NK cells expanded 2362-fold (median, range: 90-31,351) in the presence of AA and were capable of killing tumor cells under normoxia and hypoxia. Moreover, hematopoietic stem cell-derived progenitors appeared to mature faster in the presence of AA, which was also observed in the NK-cell differentiation from early T/NK-cell progenitors. CONCLUSIONS: Mature NK cells proliferate faster in the presence of phospho-L-AA, resulting in higher cell numbers with accurate functional capacity, which is required for adoptive immunotherapy.

Monitoring the initiation and kinetics of human dendritic cell-induced polarization of autologous naive CD4+ T cells.

A crucial step in generating de novo immune responses is the polarization of naive cognate CD4+ T cells by pathogen-triggered dendritic cells (DC). In the human setting, standardized DC-dependent systems are lacking to study molecular events during the initiation of a naive CD4+ T cell response. We developed a TCR-restricted assay to compare different pathogen-triggered human DC for their capacities to instruct functional differentiation of autologous, naive CD4+ T cells. We demonstrated that this methodology can be applied to compare differently matured DC in terms of kinetics, direction, and magnitude of the naive CD4+ T cell response. Furthermore, we showed the applicability of this assay to study the T cell polarizing capacity of low-frequency blood-derived DC populations directly isolated ex vivo. This methodology for addressing APC-dependent instruction of naive CD4+ T cells in a human autologous setting will provide researchers with a valuable tool to gain more insight into molecular mechanisms occurring in the early phase of T cell polarization. In addition, it may also allow the study of pharmacological agents on DC-dependent T cell polarization in the human system.

Technical advance: ascorbic acid induces development of double-positive T cells from human hematopoietic stem cells in the absence of stromal cells.

The efficacy of donor HSCT is partly reduced as a result of slow post-transplantation immune recovery. In particular, T cell regeneration is generally delayed, resulting in high infection-related mortality in the first years post-transplantation. Adoptive transfer of in vitro-generated human T cell progenitors seems a promising approach to accelerate T cell recovery in immunocompromised patients. AA may enhance T cell proliferation and differentiation in a controlled, feeder-free environment containing Notch ligands and defined growth factors. Our experiments show a pivotal role for AA during human in vitro T cell development. The blocking of NOS diminished this effect, indicating a role for the citrulline/NO cycle. AA promotes the transition of proT1 to proT2 cells and of preT to DP T cells. Furthermore, the addition of AA to feeder cocultures resulted in development of DP and SP T cells, whereas without AA, a preT cell-stage arrest occurred. We conclude that neither DLL4-expressing feeder cells nor feeder cell conditioned media are required for generating DP T cells from CB and G-CSF-mobilized HSCs and that generation and proliferation of proT and DP T cells are greatly improved by AA. This technology could potentially be used to generate T cell progenitors for adoptive therapy.

Inflammation-restraining effects of prostaglandin E2 on natural killer-dendritic cell (NK-DC) interaction are imprinted during DC maturation.

Among prostaglandins (PGs), PGE2 is abundantly expressed in various malignancies and is probably one of many factors promoting tumor growth by inhibiting tumor immune surveillance. In the current study, we report on a novel mechanism by which PGE2 inhibits in vitro natural killer-dendritic cell (NK-DC) crosstalk and thereby innate and adaptive immune responses via its effect on NK-DC crosstalk. The presence of PGE2 during IFN-γ/membrane fraction of Klebsiella pneumoniae DC maturation inhibits the production of chemokines (CCL5, CCL19, and CXCL10) and cytokines (IL-12 and IL-18), which is cAMP-dependent and imprinted during DC maturation. As a consequence, these DCs fail to attract NK cells and show a decreased capacity to trigger NK cell IFN-γ production, which in turn leads to reduced T-helper 1 polarization. In addition, the presence of PGE2 during DC maturation impairs DC-mediated augmentation of NK-cell cytotoxicity. Opposed to their inhibitory effects on peripheral blood-derived NK cells, PGE2 matured DCs induce IL-22 secretion of inflammation constraining NKp44(+) NK cells present in mucosa-associated lymphoid tissue. The inhibition of NK-DC interaction is a novel regulatory property of PGE2 that is of possible relevance in dampening immune responses in vivo.

Klebsiella pneumoniae-triggered DC recruit human NK cells in a CCR5-dependent manner leading to increased CCL19-responsiveness and activation of NK cells.

Besides their role in destruction of altered self-cells, NK cells have been shown to potentiate T-cell responses by interacting with DC. To take advantage of NK-DC crosstalk in therapeutic DC-based vaccination for infectious diseases and cancer, it is essential to understand the biology of this crosstalk. We aimed to elucidate the in vitro mechanisms responsible for NK-cell recruitment and activation by DC during infection. To mimic bacterial infection, DC were exposed to a membrane fraction of Klebsiella pneumoniae, which triggers TLR2/4. DC matured with these bacterial fragments can actively recruit NK cells in a CCR5-dependent manner. An additional mechanism of DC-induced NK-cell recruitment is characterized by the induction of CCR7 expression on CD56(dim) CD16(+) NK cells after physical contact with membrane fraction of K. pneumoniae-matured DC, resulting in an enhanced migratory responsiveness to the lymph node-associated chemokine CCL19. Bacterial fragment-matured DC do not only mediate NK-cell migration but also meet the prerequisites needed for augmentation of NK-cell cytotoxicity and IFN-γ production, the latter of which contributes to Th1 polarization.

Surface Mucin-1 does not play a role in dendritic cell migration.

Mucin-1 (MUC1) is a transmembrane glycoprotein that is upregulated upon maturation of dendritic cells (DC) in vitro or in vivo. One of the proposed functions of surface expressed MUC1 is its involvement in migration of cells. We hypothesized that MUC1 is involved in DC migration since mature DC (mDC) are highly migratory cells and MUC1 is upregulated on the surface of DC upon maturation. In this study we cultured DC using two maturation cocktails, one cocktail containing IL-4, GM-CSF, TNFalpha, PGE2, IL-1 beta and IL-6 (TP1,6-DC) and the other IL-13, GM-CSF, Ribomunyl and IFN-gamma (RI-DC). Both maturation cocktails render DC with a similar surface phenotype including CCR7 expression, but only the former induces a migratory capacity of DC to a CCL19 gradient. To analyze the role of surface-expression of MUC1 on TP1,6-DC, that are capable of migration, expression of MUC1 was prevented by adding an anti-MUC1 antibody (Ab) during the maturation process. Compared with matured DC in the absence of the Ab, no difference was observed in chemokine-induced migratory behaviour between the MUC1+ and MUC1- DC populations in a standard Transwell chemotaxis assay, nor in organotypic cultures. Our data clearly demonstrate that surface MUC1 on DC does not influence intrinsic cell-motility, nor is it involved in cell-cell and cell-matrix dependent migration.

Cancer specific Mucin-1 glycoforms are expressed on multiple myeloma.

Present therapies cannot cure the large majority of patients with multiple myeloma (MM) and therefore new treatment strategies are imperative. This study analysed the different glycosylation profiles of Mucin-1 (MUC1) on MM and acute myeloid leukaemia (AML) cells using a series of anti-MUC1 antibodies. Seventy-three per cent of the MM patients had plasma cells that expressed the fully glycosylated forms of MUC1. In contrast to controls, normal bone marrow cells and AML cells, the differentiation-dependent and cancer-associated glycoforms of MUC1 were present on 59% and 36% MM tumour cells respectively. This indicated that aberrantly glycosylated MUC1 is a potential immunotherapeutic target in MM patients.

Mucin-1 is expressed on dendritic cells, both in vitro and in vivo.

Dendritic cells (DCs) are the best professional antigen-presenting cells to stimulate cytotoxic as well as T helper cells and are therefore appropriate candidates for establishing immunotherapy. The concept of our vaccination program is to introduce the tumor-associated antigen mucin-1 (MUC1) into DCs. Analysis of immature and mature DCs--before transducing the antigen MUC1--already demonstrated expression of MUC1 on in vitro monocyte-derived DCs upon maturation. Different culture methods as well as maturation cocktails showed similar results concerning the upregulation of MUC1 expression. Furthermore, we studied the expression of MUC1 on DCs in vivo. No MUC1 expression was found on blood DCs, or on thymic or tonsil DCs. On the other hand, synovial fluid from patients with arthritis contained DCs that were found to express MUC1. This study shows for the first time that the tumor-associated antigen MUC1 is expressed on in vivo DCs. We further show that MUC1 is also expressed on in vitro cultured bone marrow-derived DCs of human MUC1 transgenic mice, supporting the relevance of this mouse model to the human situation. The observation that MUC1 is present on in vivo DCs suggests a functional role, but this physiological function remains to be elucidated.