Deficiency of SARS-CoV-2 T-cell responses after vaccination in long-term allo-HSCT survivors translates into abated humoral immunity

Recipients of allogeneic hematopoietic stem cell transplantation (allo-HSCT) for hematological diseases are at risk of severe disease and death from COVID-19. To determine the safety and immunogenicity of BNT162b2 and mRNA-1273 COVID-19 vaccines, samples from 50 infection-naive allo-HSCT recipients (median, 92 months from transplantation, range, 7-340 months) and 39 healthy controls were analyzed for serum immunoglobulin G (IgG) against the receptor binding domain (RBD) within spike 1 (S1) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; anti–RBD-S1 IgG) and for SARS-CoV-2–specific T-cell immunity, reflected by induction of T-cell–derived interferon-g in whole blood stimulated ex vivo with 15-mer SI-spanning peptides with 11 amino acid overlap S1-spanning peptides. The rate of seroconversion was not significantly lower in allo-transplanted patients than in controls with 24% (12/50) and 6% (3/50) of patients remaining seronegative after the first and second vaccination, respectively. However, 58% of transplanted patients lacked T-cell responses against S1 peptides after 1 vaccination compared with 19% of controls (odds ratio [OR] 0.17; P 5 .009, Fisher’s exact test) with a similar trend after the second vaccination where 28% of patients were devoid of detectable specific T-cell immunity, compared with 6% of controls (OR 0.18; P 5 .02, Fisher’s exact test). Importantly, lack of T-cell reactivity to S1 peptides after vaccination heralded substandard levels (,100 BAU/mL) of anti–RBD-S1 IgG 5 to 6 months after the second vaccine dose (OR 8.2; P 5 .007, Fisher’s exact test). We conclude that although allo-HSCT recipients achieve serum anti–RBD-S1 IgG against SARS-CoV-2 after 2 vaccinations, a deficiency of SARS-CoV-2–specific T-cell immunity may subsequently translate into insufficient humoral responses

NOX2-Derived Reactive Oxygen Species in Cancer

The formation of reactive oxygen species (ROS) by the myeloid cell NADPH oxidase NOX2 is critical for the destruction of engulfed microorganisms. However, recent studies imply that ROS, formed by NOX2+ myeloid cells in the malignant microenvironment, exert multiple actions of relevance to the growth and spread of neoplastic cells. By generating ROS, tumor-infiltrating myeloid cells and NOX2+ leukemic myeloid cells may thus (i) compromise the function and viability of adjacent cytotoxic lymphocytes, including natural killer (NK) cells and T cells, (ii) oxidize DNA to trigger cancer-promoting somatic mutations, and (iii) affect the redox balance in cancer cells to control their proliferation and survival. Here, we discuss the impact of NOX2-derived ROS for tumorigenesis, tumor progression, regulation of antitumor immunity, and metastasis. We propose that NOX2 may be a targetable immune checkpoint in cancer

Impact of IL-1β and the IL-1R antagonist on relapse risk and survival in AML patients undergoing immunotherapy for remission maintenance

International audienceInterleukin-1 beta (IL-1β), a pro-inflammatory cytokine, has been ascribed a role in the expansion of myeloid progenitors in acute myeloid leukemia (AML) and in promoting myeloid cell-induced suppression of lymphocyte-mediated immunity against malignant cells. This study aimed at defining the potential impact of IL-1β in the post-remission phase of AML patients receiving immunotherapy for relapse prevention in an international phase IV trial of 84 patients (ClinicalTrials.gov; NCT01347996). Consecutive serum samples were collected from AML patients in first complete remission (CR) who received cycles of relapse-preventive immunotherapy with histamine dihydrochloride (HDC) and lowdose interleukin-2 (IL-2). Low IL-1β serum levels before and after the first HDC/IL-2 treatment cycle favorably prognosticated leukemia-free survival and overall survival. Serum levels of IL-1β were significantly reduced in patients receiving HDC/IL-2. HDC also reduced the formation of IL-1β from activated human PBMCs in vitro. Additionally, high serum levels of the IL-1 receptor antagonist IL-1RA were associated with favorable outcome, and AML patients with low IL-1β along with high IL-1RA levels were strikingly protected against leukemic relapse. Our results suggest that strategies to target IL-1β might impact on relapse risk and survival in AML

Anti-Leukemic Properties of Histamine in Monocytic Leukemia: The Role of NOX2

In patients with acute myeloid leukemia (AML), treatment with histamine dihydrochloride (HDC) and low-dose IL-2 (HDC/IL-2) in the post-chemotherapy phase has been shown to reduce the incidence of leukemic relapse. The clinical benefit of HDC/IL-2 is pronounced in monocytic forms of AML, where the leukemic cells express histamine type 2 receptors (H2R) and the NAPDH oxidase-2 (NOX2). HDC ligates to H2Rs to inhibit NOX2-derived formation of reactive oxygen species, but details regarding the anti-leukemic actions of HDC remain to be elucidated. Here, we report that human NOX2+ myelomonocytic/monocytic AML cell lines showed increased expression of maturation markers along with reduced leukemic cell proliferation after exposure to HDC in vitro. These effects of HDC were absent in corresponding leukemic cells genetically depleted of NOX2 (NOX2−/−). We also observed that exposure to HDC altered the expression of genes involved in differentiation and cell cycle progression in AML cells and that these effects required the presence of NOX2. HDC promoted the differentiation also of primary monocytic, but not non-monocytic, AML cells in vitro. In a xenograft model, immunodeficient NOG mice were inoculated with wild-type or NOX2−/− human monocytic AML cells and treated with HDC in vivo. The administration of HDC reduced the in vivo expansion of NOX2+/+, but not of NOX2−/− human monocytic AML cells. We propose that NOX2 may be a conceivable target in the treatment of monocytic AML

FIGURE 4 from Impact of Surgery-Induced Myeloid-derived Suppressor Cells and the NOX2/ROS Axis on Postoperative Survival in Human Pancreatic Cancer

M-MDSC suppress NK cells by generating ROS and show enhanced NK cell suppression after surgery. A–C, NK cells were co-cultured with or without PANC-1 cells and freshly isolated healthy donor (HD) monocytes in the presence or absence of 100 µmol/L HDC, 200 U/mL catalase (Cat), 10 µmol/L GSK2795039 (GSK) or 1% DMSO (control). NK cell activation and degranulation were measured by flow cytometry and are shown by frequencies of NK cells expressing CD69+CD16+ (A) or CD107a+CD16+ (B), respectively. C, NK cell cytotoxicity against PANC-1 cells was determined by frequency of LIVE/DEAD stained PANC-1 cells. NK cell cytotoxicity against PANC-1 cells was measured in four independent experiments, that each included two to three combinations of NK cells and monocyte from different donors. D, ROS formation was measured by CL from CD33+ cells isolated 3–5 days after surgery in the absence of stimulation (PDAC Ctrl, orange), in the presence of rituximab (PDAC RTX, red), in the presence of rituximab and HDC (PDAC RTX + HDC, green) and from 2 HDs stimulated with rituximab (mean ROS formation shown, HD RTX, black). NK cells were cocultured with K562 cells and CD33+ cells isolated from HDs or patients before surgery (pre-op) or 3–5 days after surgery (days 3–5) in the presence (HDC) or absence of HDC. NK cell activation and degranulation were measured by flow cytometry as frequencies of CD16+ NK cells expressing CD69 (E) or CD107a (F) in NK:K562:Myeloid cell cocultures. NK cell cytotoxicity was measured in 10 independent experiments that were normalized by setting the mean NK cell cytotoxicity against K562 in the absence of myeloid cells to 1 in each experiment. Statistics by mixed-effects analysis followed by Šídák multiple comparison test (P-values indicated by *). HD and cancer samples were compared using Kruskal–Wallis test followed by Dunn multiple comparison test (P-values indicated by #).*, P /##, P P P < 0.0001.</p

Table S1 from Impact of Surgery-Induced Myeloid-derived Suppressor Cells and the NOX2/ROS Axis on Postoperative Survival in Human Pancreatic Cancer

Sequences of primers used for RT-PCR</p

FIGURE 5 from Impact of Surgery-Induced Myeloid-derived Suppressor Cells and the NOX2/ROS Axis on Postoperative Survival in Human Pancreatic Cancer

NOX2 inhibition prevents surgery-induced metastasis in a murine model. A, Schematic outline of the experimental model of surgery-induced metastasis. Blood samples were collected from naïve (Ctrl) and sponge-bearing (Inf) WT and NOX2-KO mice 1 week after a surgical procedure (sponge implantation). A group of WT mice received intraperitoneal injections with histamine dihydrochloride (WT HDC) every other day starting one day before surgery and continuing until 3 days after tumor cell challenge. Frequency of CD11b+Ly6C+M-MDSC (B) and intracellular ROS levels in M-MDSC (C) were measured by flow cytometry. At 8 days postsurgery, mice were intravenously inoculated with B16F10 cells. D, Metastatic lesions in lungs were enumerated 19–21 days after tumor cell challenge. Differences in inflammatory monocytes, DCFDA levels, and tumor numbers in vivo were calculated using one-way ANOVA following Holm-Šídák multiple comparisons test. The frequency of M-MDSC and number of metastatic foci was evaluated in four independent experiments for WT mice with or without surgery-induced inflammation, and in two experiments for KO mice and HDC treated mice. DCFDA levels of M-MDSC were measured in three independent experiments for WT mice, two experiments for KO mice, and one experiment for HDC-treated mice. In each experiment, 4–5 mice per group were included. Fold change was calculated by dividing raw data with the mean of raw data in the WT mice control group in each experiment. *, P P P P < 0.0001.</p

FIGURE 1 from Impact of Surgery-Induced Myeloid-derived Suppressor Cells and the NOX2/ROS Axis on Postoperative Survival in Human Pancreatic Cancer

Pancreatic cancer surgery triggers inflammation and expansion of M-MDSC. A, Flow chart of included IPEP study participants. B, Study design and available samples. Absolute numbers of neutrophils (C), monocytes (D), and lymphocytes (E) in patients undergoing surgery for periampullary cancer with whole blood samples (differential counts) analyzed before (pre-op) and 1, 3–5 and 28 days after pancreatic cancer surgery (day 1, days 3–5, and day 28, respectively; N = 17 for pre-op, N = 16 for day 1, N = 15 for days 3–5, N = 11 for day 28). F, Plasma levels of IL6 in healthy donors (HD) and in pre- and post-op samples from patients (N = 16 for pre-op and for day 1, N = 15 for days 3–5, N = 9 for day 28, N = 6 for HD). G–I, CD14+HLADRlow M-MDSC in peripheral blood of healthy donors and patients before and after surgery analyzed by flow cytometry. G, Representative FACS plot of the frequency of CD14+HLADRlow cells among CD14+ cells. Absolute numbers (H) and frequencies (I) of M-MDSC (N = 17 for pre-op, N = 16 for day 1 and days 3–5, N = 10 for day 28, N = 12 for HD). J, M-MDSC levels of ROS as measured by DCFDA MFI (N = 16 for pre-op, for day 1 and for days 3–5, N = 10 for day 28, N = 12 for HD). Preoperative and postoperative samples were compared using mixed-effects analysis followed by Šídák multiple comparison test (P-values indicated by *). HD and cancer samples were compared using Kruskal–Wallis test followed by Dunn multiple comparison test (P-values indicated by #). */#P ##P /###P /####P < 0.0001.</p

Figure S5 from Impact of Surgery-Induced Myeloid-derived Suppressor Cells and the NOX2/ROS Axis on Postoperative Survival in Human Pancreatic Cancer

DC frequences and DC marker expression before and after surgery</p

Data_Sheet_1_Anti-Leukemic Properties of Histamine in Monocytic Leukemia: The Role of NOX2.PDF

<p>In patients with acute myeloid leukemia (AML), treatment with histamine dihydrochloride (HDC) and low-dose IL-2 (HDC/IL-2) in the post-chemotherapy phase has been shown to reduce the incidence of leukemic relapse. The clinical benefit of HDC/IL-2 is pronounced in monocytic forms of AML, where the leukemic cells express histamine type 2 receptors (H₂R) and the NAPDH oxidase-2 (NOX2). HDC ligates to H₂Rs to inhibit NOX2-derived formation of reactive oxygen species, but details regarding the anti-leukemic actions of HDC remain to be elucidated. Here, we report that human NOX2⁺ myelomonocytic/monocytic AML cell lines showed increased expression of maturation markers along with reduced leukemic cell proliferation after exposure to HDC in vitro. These effects of HDC were absent in corresponding leukemic cells genetically depleted of NOX2 (NOX2^−/−). We also observed that exposure to HDC altered the expression of genes involved in differentiation and cell cycle progression in AML cells and that these effects required the presence of NOX2. HDC promoted the differentiation also of primary monocytic, but not non-monocytic, AML cells in vitro. In a xenograft model, immunodeficient NOG mice were inoculated with wild-type or NOX2^−/− human monocytic AML cells and treated with HDC in vivo. The administration of HDC reduced the in vivo expansion of NOX2^+/+, but not of NOX2^−/− human monocytic AML cells. We propose that NOX2 may be a conceivable target in the treatment of monocytic AML.</p