Heterologous prime-boost viral vector vaccines for the treatment of a P1A-expressing BGL-1 glioblastoma model

Glioblastoma is an extremely aggressive and difficult cancer to treat, which may partly be due to its limited ability to induce T-cell responses. However, combining viral vector vaccines with other therapies to generate tumor-specific T cells may provide a meaningful benefit to patients. Here, we investigated whether heterologous prime-boost vaccination with chimpanzee-derived adenoviral vector ChAdOx1 and modified vaccinia Ankara (MVA) vaccines could generate therapeutically effective CD8+ T-cell responses against a model antigen P1A, a mouse homolog of human tumorassociated Melanoma Antigen GenE (MAGE)-type antigens, expressed by a BGL-1 mouse glioblastoma cell line. We demonstrated that heterologous prime-boost vaccination with ChAdOx1/MVA vaccines targeting P1A generated a high magnitude of CD8+ T cells specific for the P1A35-43 epitope presented by the MHC class I molecule H-2Ld . Prophylactic vaccination with ChAdOx1/MVA-P1A significantly prolonged the survival of syngeneic mice subcutaneously challenged with P1A-expressing BGL-1 tumors. Furthermore, different vaccination schedules significantly impact the magnitude of antigen-specific CD8+ T-cell responses and may impact protective efficacy. However, the substantial induction of myeloid-derived suppressor cells (MDSCs) by this tumor model presents a significant challenge in the therapeutic setting. Future work will investigate the efficacy of this vaccination strategy on intracranial P1A-expressing BGL-1 models.

Cellular immune states in SARS-CoV-2-induced disease.

The general immune state plays important roles against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Cells of the immune system are encountering rapid changes during the acute phase of SARS-CoV-2-induced disease. Reduced fraction of functional CD8+ T cells, disrupted cross-talking between CD8+ T cells with dendritic cells (DCs), and impaired immunological T-cell memory, along with the higher presence of hyperactive neutrophils, high expansion of myeloid-derived suppressor cells (MDSCs) and non-classical monocytes, and attenuated cytotoxic capacity of natural killer (NK) cells, are all indicative of low efficient immunity against viral surge within the body. Immune state and responses from pro- or anti-inflammatory cells of the immune system to SARS-CoV-2 are discussed in this review. We also suggest some strategies to enhance the power of immune system against SARS-CoV-2-induced disease.

Impaired Cellular Immunity and Elevated Myeloid Derived Suppressor Cells in Kidney Transplant Recipients with Impaired Antibody Response to Covid-19 Vaccination

Purpose: To evaluate post-vaccination cellular and antibody (Ab) immunity after COVID-19 vaccination in single blood samples from 17 kidney transplant (KT) recipients who had received COVID-19 vaccination Methods: We measured frequencies of peripheral blood T- and B-cells which expressed the inflammatory marker CD154 after overnight stimulation with peptide mixtures representing the spike protein S, its S2 component which is conserved between SARS-CoV-2 and human coronaviruses, and the S1 component, which is specific to SARS-CoV-2 and also contains its receptor binding domain (RBD). Serum from each sample was assayed for anti-RBD and anti-S IgG Abs with ELISA. Optical density at 450nm (OD450) of 0.45 or greater implied presence of either Ab. Frequencies of monocytic and polymorphonuclear (PMN) myeloid-derived suppressor cell were also measured with flow cytometry. Result(s): Median age was 40 yrs (range 25 to 83), male:female gender distribution was 7:8. All recipients received mRNA vaccination. Anti-S-IgG and anti-RBD-IgG were detected in 11 (Ab+) and were absent in four (Ab-). Compared with Ab+ KT recipients, those who were Ab- had lower frequencies of S2-reactive and S-reactive B-cells (p<0.05), CD4+ and CD8+ T-cells (Table 1, Fig 1). S1-reactive T-cell and B-cells were non-detectable. Frequencies of PMN-MDSC were numerically higher in Ab- compared with Ab+ KT recipients (Mean +/- SEM 38.9+/-8.1 vs 19.4+/-1.8, p-value 0.1, NS). Significant negative correlation was observed between PMN-MDSC frequencies and strength of anti-RBD IgG and anti-SPIKE IgG (Fig 1). Conclusion(s): COVID-19 vaccination results in spike antigen reactive T- and B-cells in KT recipients who develop Abs after vaccination. Failure of an Ab response is associated with impaired B-cell responses to the spike antigen and an increase in circulating polymorphonuclear myeloid derived suppressor cells. (Table Presented).

Rapid increase of myeloid-derived suppressor cells and prolonged innate immune dysfunctions in patients with COVID-19

Background: PMN and monocytic myeloid-derived suppressor cells (PMN-MDSCs, M-MDSCs) are immunosuppressive cells rising during infections. Aim: To characterize the dynamic of MDSCs in relation with immune parameters in COVID-19 patients followed for 3 months. Methods: 56 SARS-CoV-2 infected adult patients hospitalized at CHUV were included. Blood was obtained at inclusion and 3 months later in 21 patients, and from 10 healthy controls. Blood was stimulated with TLR ligands. Leukocyte populations and cytokines were analyzed by flow cytometry, mass cytometry, multiplex bead assay and ELISA. Results: At hospital admission, PMN-MDSCs and M-MDSCs were increased 2-4-fold in COVID-19 patients (P <0.05). PMN-MDSCs and M-MDSCs counts were higher in severe than in moderate COVID-19 patients (P <0.005). PMN-MDSCs and M-MDSCs correlated positively with EGF and HGF (P <0.05). M-MDSCs correlated positively with IL-1β, IL-7, PDGF and VEGF (P <0.05). In whole blood stimulated with TLR ligands, the proportion of TNF and IL-6- producing monocytes and DCs were reduced in patients. After 32 months, MDSCs were back to normal levels, while the production of cytokines by blood, monocytes and DCs was still largely affected. Conclusions: PMN-MDSCs and M-MDSCs were elevated and correlated with disease severity in patients analyzed at hospitalization. Innate immune blood responses were impaired in patients, which persisted for up to 3 months. Our results suggest that COVID-19 induces rapid and long-standing innate immune dysregulation.

Redirection of anti-virus immunity towards anti-tumor immunity using nanoparticle-induced immune tolerance for oncolytic adenovirus therapy

Recent clinical observations that some coronavirus infections induced complete remissions in lymphoma patients emphasized again the potential of cancer virotherapy. Infection of cancer cells with oncolytic viruses reshapes the tumor microenvironment by activating anti-viral and anti-tumor immunity. A phase 1 clinical trial using oncolytic adenovirus Delta-24-RGD (DNX-2401) to treat recurrent malignant gliomas demonstrated activation of CD8+ T-cells and significant clinical benefits for a subset of patients. However, both anti-virus and anti-tumor immune responses are contingent on the activation of respective clones of CD8+ T-cells, which compete for clonal expansion. Thus, overexpansion of T-cells against viral antigens reduces the frequency of subdominant clones against tumor antigens. We hypothesized that inducing immune tolerance for viral antigens will decrease anti-viral immunity and in turn derepress anti-tumor immunity, resulting in enhanced efficacy of cancer virotherapy. In this work, we used nanoparticles encapsulating adenoviral antigens E1A, E1B and hexon that distributed to liver resident macrophages (P<0.0001) and induced peripheral immune tolerance. Functional experiments to restimulate immune cells with viral or tumor antigens showed that injection of nanoparticles induced virus-specific immune tolerance and redirected the focus of the immune response towards tumor peptides as measured by interferon-gamma secretion (P<0.0001). Co-culture experiments also showed increased activation of immune cells against fixed tumor cells after nanoparticle treatment (P<0.0001). Reduction of virus-specific T-cells and concurrent expansion of tumor-specific T-cell clones were further confirmed with E1A or OVA tetramers (P<0.05). Flow cytometry analysis suggested increased anti-tumor responses were due to differences in T-cell clones and not due to other immune populations including natural killer cells or myeloid-derived suppressor cells (P=0.3). Importantly, virotherapy in combination with nanoparticle-induced immune tolerance towards viral antigens in tumor-bearing mice increased the overall survival and doubled the percentage of long-term survivors compared to virus treatment alone. Our data should propel the development of a future clinical trial aiming to maximize the potential of anti-tumor immunity during cancer virotherapies.

Myeloid-Derived Suppressor Cells and Clinical Outcomes in Children With COVID-19.

Background: Although children with COVID-19 account for fewer hospitalizations than adults, many develop severe disease requiring intensive care treatment. Critical illness due to COVID-19 has been associated with lymphopenia and functional immune suppression. Myeloid-derived suppressor cells (MDSCs) potently suppress T cells and are significantly increased in adults with severe COVID-19. The role of MDSCs in the immune response of children with COVID-19 is unknown. Aims: We hypothesized that children with severe COVID-19 will have expansion of MDSC populations compared to those with milder disease, and that higher proportions of MDSCs will correlate with clinical outcomes. Methods: We conducted a prospective, observational study on a convenience sample of children hospitalized with PCR-confirmed COVID-19 and pre-pandemic, uninfected healthy controls (HC). Blood samples were obtained within 48 h of admission and analyzed for MDSCs, T cells, and natural killer (NK) cells by flow cytometry. Demographic information and clinical outcomes were obtained from the electronic medical record and a dedicated survey built for this study. Results: Fifty children admitted to the hospital were enrolled; 28 diagnosed with symptomatic COVID-19 (10 requiring ICU admission) and 22 detected by universal screening (6 requiring ICU admission). We found that children with severe COVID-19 had a significantly higher percentage of MDSCs than those admitted to the ward and uninfected healthy controls. Increased percentages of MDSCs in peripheral blood mononuclear cells (PBMC) were associated with CD4+ T cell lymphopenia. MDSC expansion was associated with longer hospitalizations and need for respiratory support in children admitted with acute COVID-19. Conclusion: These findings suggest that MDSCs are part of the dysregulated immune responses observed in children with severe COVID-19 and may play a role in disease pathogenesis. Future mechanistic studies are required to further understand the function of MDSCs in the setting of SARS-CoV-2 infection in children.

Exploring the Role of Low-Density Neutrophils During Mycobacterium tuberculosis Infection.

Tuberculosis (TB) is caused by infection with the bacterium Mycobacterium tuberculosis (Mtb), which primarily infects the lungs but can also cause extrapulmonary disease. Both the disease outcome and the pathology of TB are driven by the immune response mounted by the host. Infection with Mtb elicits inflammatory host responses that are necessary to control infection, but can also cause extensive tissue damage when in excess, and thus must be precisely balanced. In particular, excessive recruitment of neutrophils to the site of infection has been associated with poor control of Mtb infection, prompting investigations into the roles of neutrophils in TB disease outcomes. Recent studies have revealed that neutrophils can be divided into subpopulations that are differentially abundant in TB disease states, highlighting the potential complexities in determining the roles of neutrophils in Mtb infection. Specifically, neutrophils can be separated into normal (NDN) and low-density neutrophils (LDNs) based on their separation during density gradient centrifugation and surface marker expression. LDNs are present in higher numbers during active TB disease and increase in frequency with disease progression, although their direct contribution to TB is still unknown. In addition, the abundance of LDNs has also been associated with the severity of other lung infections, including COVID-19. In this review, we discuss recent findings regarding the roles of LDNs during lung inflammation, emphasizing their association with TB disease outcomes. This review highlights the importance of future investigations into the relationship between neutrophil diversity and TB disease severity.

Myeloid-derived suppressor cells in COVID-19: A review.

Coronavirus disease 2019 (COVID-19) is a potentially life-threatening infection characterized by excessive inflammation, coagulation disorders and organ damage. A dysregulated myeloid cell compartment is one of the most striking immunopathologic signatures of this newly emerged infection. A growing number of studies are reporting on the expansion of myeloid cells with immunoregulatory activities in the periphery and airways of COVID-19 patients. These cells share phenotypic and functional similarities with myeloid-derived suppressor cells (MDSCs), which were first described in cancer patients. MDSCs are a heterogeneous population of pathologically activated myeloid cells that exert immunosuppressive activities against mainly effector T cells. The increased frequency of these cells in COVID-19 patients suggests that they are involved in immune regulation during this infection. In this article, we review the current findings on MDSCs in COVID-19 and discuss the complex role of these cells in the immunopathology of COVID-19.

Sars-Cov-2 T-Cell Response in Allogeneic Hematopoietic Stem Cell Recipients Following Two Doses of BNT162b2 Vaccine

Introduction: Virus-specific humoral and cellular immune responses act synergistically to protect from viral infection. In our recent observational monocentric study of 117 hematopoietic stem cell adult recipients, we found that 54% and 83 % patients achieved a humoral response after two doses of BNT162b2 anti-SARS-CoV-2 messenger RNA vaccine (Pfizer BioNTech), respectively. Here, we evaluated the T-cell response against the SARS-Cov-2 spike protein after two doses of BNT162b2 vaccine in some allografted patients from the same cohort and compared these results to those from healthy controls. Methods: To quantify SARS-CoV-2 specific T-cells, we used an INFg ELISpot assay that detects these cells after activation of peripheral blood mononuclear cells (PBMC) with 3 peptide pools covering the whole protein sequence of the spike glycoprotein (Prot _S1;_S+ and _S PepTivator peptide pools, Miltenyi Biotec, Bergisch Gladbach, Germany). EBV and CMV specific T-cells were also quantified as controls. The immunophenotype of PBMC was determined by flow cytometry, after dead cell exclusion, with monoclonal antibodies identifying the following surface antigens: CD45, CD3, CD14, CD19 and HLA-DR. The frequencies of spot-forming units (SFU) were reported as per 10 6 CD3+ T-cells. Results: Samples from 46 allografted patients (acute myeloblastic leukemia, N=27, myelodysplastic syndrome, N=19) and 16 healthy controls were available. Characteristics of the population are given in Table 1. All fully vaccinated healthy donors became seropositive and developed a positive T-cell response to spike peptide pools even though variable frequencies were observed. The median response was 195 SFU/10 6 T-cells. By comparison, the frequency of EBV-specific T-cells was 774 SFU/10 6 T-cells (Figure 1). In the group of patients, 78% (n=36/46) had achieved a humoral response after the second dose of vaccine. Among these humoral responders (HR), 89% (n=32/36) also had a positive anti-spike T-cell response with variable frequencies (median =119 SFU/10 6 T-cells. For 8 patients, this T cell response was higher than that of controls (>800 SFU/10 6 T-cells) (Figure 1), which is equivalent to more than 1 specific T-cell per microliter of blood (Figure 2). The humoral responders (HR) who did not develop a T-cell response (11%, n=4/36) had a median time from transplant to vaccination of 523 days compared to 1032 days for cellular responder patients. Among the 10 patients who were non humoral responders (NHR) (22%, n=10/46), 4 (40%) developed a cellular immunity, including one with a very high T cell response (1333 SFU/10 6 T-cells). As expected, the absence of humoral response was observed in patients who were within one year of the transplant. Of note, somehow unexpectedly, patients often presented a high frequency of EBV- and CMV-specific T cells (Figures 1 & 2). As expected, PBMC immunophenotypic analysis revealed that CD3+ frequencies were lower in patients compared to those of controls but were similar between HR and NHR. NHR had very low frequencies of B cells and interestingly, they had an elevated frequency of CD14+ monocytes with low/neg HLA-DR expression potentially corresponding to myeloid-derived suppressor cells (MDSCs) (Figure 3). Conclusion: In this series, 89% of allografted patients who developed an anti-spike humoral response also presented an anti-SARS-Cov-2 cellular immunity. Interestingly, anti-SARS-Cov-2 specific T-cells could be detected in 40% of NHR patients. Although a larger group of patients is required to confirm these results, it remains to be determined whether this T-cell response is protective against SARS-Cov-2 infection as previously demonstrated for CMV (Litjens et al, 2017). Finally, the role of potential immunosuppressive MDSCs must be explored in patients who develop no sign of T-cell response after vaccination. [Formula presented] Disclosures: Moreau: Oncopeptides: Honoraria;Celgene BMS: Honoraria;Sanofi: Honoraria;Abbvie: Honoraria;Janssen: Honoraria;Amgen: Honoraria.

The Enigma of Low-Density Granulocytes in Humans: Complexities in the Characterization and Function of LDGs during Disease.

Low-density granulocytes (LDGs) have been characterized as important immune cells during healthy and disease states in humans, including microbial infections, cancer, and autoimmune dysfunction. However, the classification of this cell type is similar to other immune cells (e.g., neutrophils, myeloid-derived suppressor cells) and ambiguous functional standards have rendered LDG identification and isolation daunting. Furthermore, most research involving LDGs has mainly focused on adult cells and subjects, leaving increased uncertainty surrounding younger populations, especially in vulnerable neonatal groups where LDG numbers are elevated. This review aims to bring together the current research in the field of LDG biology in the context of immunity to disease, with a focus on infection. In addition, we propose to highlight the gaps in the field that, if filled, could improve upon isolation techniques and functional characterizations for LDGs separate from neutrophils and myeloid-derived suppressor cells (MDSCs). This will not only enhance understanding of LDGs during disease processes and how they differ from other cell types but will also aid in the interpretation of comparative studies and results with the potential to inform development of novel therapeutics to improve disease states in patients.

Myeloid cell dynamics correlating with clinical outcomes of severe COVID-19 in Japan.

An expanded myeloid cell compartment is a hallmark of severe coronavirus disease 2019 (COVID-19). However, data regarding myeloid cell expansion have been collected in Europe, where the mortality rate by COVID-19 is greater than those in other regions including Japan. Thus, characteristics of COVID-19-induced myeloid cell subsets remain largely unknown in the regions with low mortality rates. Here, we analyzed cellular dynamics of myeloid-derived suppressor cell (MDSC) subsets and examined whether any of them correlate with disease severity and prognosis, using blood samples from Japanese COVID-19 patients. We observed that polymorphonuclear (PMN)-MDSCs, but not other MDSC subsets, transiently expanded in severe cases but not in mild or moderate cases. Contrary to previous studies in Europe, this subset selectively expanded in survivors of severe cases and subsided before discharge, but such transient expansion was not observed in non-survivors in Japanese cohort. Analysis of plasma cytokine/chemokine levels revealed positive correlation of PMN-MDSC frequencies with IL-8 levels, indicating the involvement of IL-8 on recruitment of PMN-MDSCs to peripheral blood following the onset of severe COVID-19. Our data indicate that transient expansion of the PMN-MDSC subset results in improved clinical outcome. Thus, this myeloid cell subset may be a predictor of prognosis in cases of severe COVID-19 in Japan.