Multiplex measurements of the impacts of ACE2 sequence and cell surface abundance during SARS-like coronavirus entry

SARS-like coronaviruses, including SARS-CoV and SARS-CoV-2, encode spike proteins that bind human ACE2 protein on the cell surface to enter target cells and cause infection. The efficiency of virus entry depends on ACE2 sequence and expression levels in target cells. A small fraction of humans encodes variants of ACE2, thus altering the biochemical properties at the protein interaction interface. All humans possess cells with vastly differing amounts of ACE2 on the cell surface, ranging from cell types with high expression in the gut and lungs to lower expression in the liver and pancreas. Mastering our understanding of spike-ACE2 interaction and infection requires experiments precisely perturbing both variables. Thus, we developed a synthetic cell engineering approach compatible with high throughput assays for pseudo-typed virus infection. Our assay system is capable of assessing both variables individually and in combination. We adapted an engineered HEK293T DNA recombinase landing pad cell line capable of expressing transgenic ACE2 sequences at highly precise levels. Infection with lentiviruses pseudotyped with the spikes of SARS-like coronaviruses revealed that high ACE2 abundance could mask the effects of impaired binding thereby making it challenging to know the role of affinity altering mutations during infection. We limited the ACE2 abundance on the cell surface by expressing transgenic ACE2 behind a suboptimal Kozak sequence, thereby altering its protein translation rate. This allowed us to understand how ACE2 sequence could impact its interaction with coronavirus spike proteins as two human ACE2 variants at the binding interface, K31D and D355N, exhibited reduced infection. Our experiments suggested that we need to better understand how ACE2 expression determines the susceptibility of cells for SARS-like coronavirus binding and infection. We thus created an ACE2 Kozak library consisting of ~4,096 Kozak variants, each conferring a different ACE2 protein translation rate thus resulting in a range of ACE2 steady-state abundances. Combining fluorescence-activated cell sorting and high-throughput DNA sequencing (FACS-seq) revealed the library to span two orders of magnitude of ACE2 abundance. Challenging this library of cells with spike pseudotyped lentiviruses revealed how ACE2 abundance correlated with infection rate. The library-based experiments yielded a dynamic range wider than traditional single sample infection assay, likely more representative of infection dynamics in vivo. Now that we have characterized the impacts of ACE2 abundance on infectivity in engineered cells, our next goal is to expand the comparison to physiologically relevant cells with endogenously expressed proteins. Modulating protein abundance levels will be key to creating maximally informative functional assays for any protein in cell-based assays, and we have laid the groundwork for being able to simultaneously test the impacts of protein abundance and sequence in combination for proteins involved in diverse cellular processes. This research was supported by a National Institute of Health (NIH) grant GM142886 (KAM).Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

Evaluation of the immunogenicity of an inactivated vaccine prototype against SARS-COV- 2 virus in vitro

Background: Belarus started developing a vaccine against SARS-CoV- 2 in 2021. The aim of the first stage of investigation was to evaluate the immunogenicity of the vaccine prototypes (VP) in vitro. Method(s): SARS-CoV- 2 strains (n = 7) were isolated using Vero E6 cells, inactivated with beta-propiolactone and purified. Antigens (Ag) were adsorbed on adjuvants: Al(OH)3 (Ag+AH group) or Al3PO4 (Ag+AP group). The single dose of VP (500 ul) was composed of 10 mug of Ag adsorbed on adjuvants (200 mug of Al3+). Blood samples from SARS-CoV- 2 recovered donors (n = 18) and healthy controls having no history of COVID-19 infection (n = 5) were used. Whole blood and Tag-it Violet labeled PBMCs were cultivated with VP, pure Ag, adjuvants (0.25-1 mug of Ag, 20 mug of Al3+ for probe) or pool of peptides, covering sequence of SARS-CoV- 2 N, S, M-proteins (PP), for 6 h and 7 days respectively. INF-gamma production and proliferation of CD3+ T-cells were assayed by FACS. Result(s): Counts of CD3+IFN-gamma+ T cells were 3.14(2.72-5.13)/ 1x105 CD3+ T cells in negative control (NC), and 12.73(10.09-33.95)/ 1x105 CD3+ T cells in specific positive control (PP) (n = 18, p < 0.0001), proving presence of antigen-specific T cells (ASCs) in donor blood. Samples were considered positive for VP and Ag immunogenicity when numbers of CD3+IFN-gamma+ T cells were 1.5 times greater compared with NC. Both VP types (Ag+AP, Ag+AH) and pure SARS-CoV- 2 isolates stimulated the production of INF-gamma by ASCs, responses ranged from 1 to 4 isolates of 7 studied per donor. Immunogenicity of Ag+AP, Ag+AH was confirmed by proliferation assay. Proliferation level was 1.07(0.97-2.38)% in Ag group with no differences from NC (n = 7, p > 0.5). Proliferation was significantly greater in VP groups compared with Ag: 2.47(1.65-2.68)% in Ag+AH, 4.03(2.56-4.61)% in Ag+AP (p = 0.009 and 0.002, respectively), stimulation of T cell was stronger by Ag+AP compared with Ag+AH (p = 0.009, M-U test). Pure adjuvants did not induce T cells response. There was no T-cell stimulation by Ag and VP in samples obtained from COVID-19 negative donors. Conclusion(s): The VP against SARS-CoV- 2 infection composed of inactivated virus adsorbed on Al(OH)3 and Al3PO4 adjuvants has immunogenic properties proven in two different immunological assays. VP stimulated activation and proliferation of ASCs in vitro suggesting this VP can be used for further preclinical in vivo evaluation.

Bronchoalveolar lavage fluid characteristics in patients with persistent symptoms following COVID-19 infection

A significant number of patients suffer from persistent symptoms following COVID-19 infection. However, data regarding the immunopathological mechanisms and potential biomarkers are limited. We investigated the differential cell count in the bronchoalveolar lavage fluid (BALF) in a post-COVID cohort. Patients presenting at the Vienna General Hospital within six months of a COVID-19 infection were enrolled. All patients underwent pulmonary function tests (PFT) and low-dose HRCT at baseline and at 6 and 12 months after a COVID-19 infection. Patients with pathological findings on HRCT or impairment in PFT were offered a bronchoscopy with BALF differential cell count via FACS analysis. Out of the 305 patients enrolled, 29 underwent bronchoscopy with bronchoalveolar lavage. After a median of 84 days following initial diagnosis of COVID-19, 25 showed persistent symptoms including dyspnoea (62.1%), fatigue (10.3%) and chest pain (10.3%). 24 patients showed pathological findings on HRCT consistent with COVID-19. While 11 patients developed a restrictive lung disease defined as TLC < LLN, 18 patients showed a reduced diffusion capacity defined as DLCO < 80%. Differential cell counts revealed that some patients showed lymphocytosis (7/29), increased eosinophil counts (5/29) and elevated neutrophils counts (2/29) in the BALF. Our preliminary data show that 34.5% of patients with persistent changes on HRCT have an elevated immune cell count with lymphocytosis being the predominant pattern. The degree of alveolar lymphocytosis might correlate with the severity of restrictive lung disease and might facilitate treatment decisions in patients with persistent symptoms following COVID-19.

Delayed immunological reaction of Human Microvascular Lung Endothelium in response to Human Coronavirus 229E infection

Introduction: In the natural conditions first and major target for respiratory viruses (RVs) are epithelial cells. Nonetheless, recently we have demonstrated that RVs are able to infect not only epithelium, but also Human Microvascular Lung Endothelial Cells (HMVEC-L) which increased network is observed during severe asthma due to increased angiogenesis. Furthermore, on the surface of HMVEC-L we observed intense expression of aminopeptidase N (AP-N)- an entry receptor for Human Coronavirus 229E (HCoV-229E). Due to the facts, that possibility of being infected by HCoV-229E should be considered and there is no research based on this model the aim of this study was to assess the vulnerability of HMVEC-L to HCoV-229E infection. Method(s): HMVEC-L was incubated with HCoV-229E (MOI 0,1;1,0;3,0) for 3 hours, 3x PBS washed and cultured for 120 hours. In relevant time points (5;24;48;72;96 and 120h) viral copy number and mRNA expression of inflammatory, anti-viral and receptor factors were evaluated in Real-Time PCR. Confocal microscopy (CM) and flow cytometry (FACS) were used to measure AP-N surface expression. Result(s): FACS and CM confirmed intense surface expression of AP-N on HMVEC-L. HCoV-229E efficiently infected HMVEC-L (604 945,5 +/-194 930,2 viral copies/mul) in 48h cultures (MOI 0,1) and induced relatively late (between 72- 96h) mRNA expression of RANTES (1181,12);IL-6 (89,6);IFN-beta (53,7);OAS-1 (64,3);PKR (11,4) and TLR-3 (42,4). Increased mRNA expression was also accompanied by protein release to the supernatants. Conclusion(s): HCoV-229E may efficiently infect HMVEC-L and induce delayed inflammatory and anti-viral response.

INHIBITION OF PROTEIN KINASE CK2 AND BCL-2: A NOVEL THERAPEUTIC STRATEGY TO ANTAGONIZE MANTLE CELL LYMPHOMA CELL GROWTH

Background: Mantle cell lymphoma (MCL) is a B-cell tumor which often relapses. BCR inhibitors (Ibrutinib, Acalabrutinib) and antiapoptotic BCL2-family members blockers BH3-mimetics (Venetoclax, ABT-199) are effective drugs to fight MCL. However, the disease remains incurable, due to therapy resistance, even to the promising Venetoclax and Ibrutinib combination. Therefore, there is a profound need to explore novel useful therapeutic targets. CK2 is a S/T kinase overexpressed in several solid and blood tumors. We demonstrated that CK2, operating through a 'non-oncogene addiction' mechanism promotes tumor cell survival, and counteracts apoptosis, by activating pro-survival signaling cascades, such as NF-κ B, STAT3 and AKT. CK2 could regulate also BCL2 family members. The CK2 chemical inhibitor CX-4945 (Silmitasertib, Sil) is already under scrutiny in clinical trials in relapsed multiple myeloma, solid tumors and COVID-19. Aims: In this work, we tested the effect of CK2 chemical inhibition or knock down on Venetoclax (Ven)-induced cytotoxicity in MCL pre-clinical models to effectively reduce MCL cell growth and clonal expansion. Methods: CK2 expression and BCR/BCL2 related signaling components were analyzed in MCL cells and control cells by Western blotting. CK2 and BCL2 inhibition was obtained with Sil and Ven, respectively and with CK2 gene silencing through the generation of anti-CK2 shRNA IPTG-inducible MCL cell clones. Survival, apoptosis, mitochondrial membrane depolarization and proliferation were investigated by FACS analysis of AnnexinV/PI and JC-10 staining. The synergic action of Ven and Sil was analyzed by the Chou-Talalay combination index (CI) method. CK2 knock down in vivo was obtained in xenograft NOD-SCID mouse models Results: CK2 inactivation (with Sil or CK2 silencing) determined a reduction in the activating phosphorylation of S529 p65/RelA and S473 and S129 AKT, important survival cascades for MCL. Sil or CK2 silencing caused BCL2 and related MCL1 protein reduction, causing cell death. Importantly, we confirmed these results also in an in vivo xenograft mouse model of CK2 knockdown in MCL. Sil +Ven combination increased MCL cell apoptosis, as judged by the augmented frequency of Annexin V positive cells and expression of cleaved PARP protein, and JC-10 mitochondrial membrane depolarization, with respect to the single treatments. Captivatingly, Sil or CK2 gene silencing led to a substantial reduction of the Ven-induced increase of MCL-1, potentially counteracting a deleterious Ven-induced drawback. Analysis of cell cycle distribution confirmed an increased frequency of apoptotic cells in the sub G1 phase in CK2-silenced cells and a modulation of the other phases of the cell cycle. Remarkably, the calculated CI less than 1 suggested a strong synergic cell-killing effect between Sil and Ven, on all the cell lines tested, including those less sensitive or resistant to Ven Summary/Conclusion: We demonstrated that the simultaneous inhibition/knock down of CK2 and BCL2 synergistically cooperates in inducing apoptosis and cell cycle arrest of MCL malignant B-lymphocytes and has the potential of reducing MCL clonal growth, also counterbalancing mechanism of resistance that may arise with Ven. Therefore, CK2 is a rational therapeutic target for the treatment of MCL to be tested in combination with Ibrutinib or Ven.

Myeloid-Dependent Immunosuppressive Features Drive and Predict IPF Progression

Rationale: Despite the availability of pharmacologic therapies, idiopathic pulmonary fibrosis (IPF) is still a clinical challenge with several unmet needs. Robust evidence supports monocytes as cellular biomarkers of progression in IPF. Yet, their precise role and whether specific subtypes might predict progression and drive disease is unknown. We reported, for the first time, that myeloidderived suppressor cells (MDSC), immature precursors of monocytes, are increased in numbers, functionally active in IPF. Monocytic MDSC is the predominant subtype in IPF, and yet, functional characterization and immune modulation properties have not been explored. Methods and Results: characterization of circulating myeloid populations in IPF by multicolor FACS confirmed the abundance of MDSC (Lin-, HLA-DRlo, CD33+, CD14+, S100A+, CD28L1+ and ICOSL+) in IPF (n=78) and fILD (n=83), also abundant in whole blood scRNA seq of severe Covid-19 patients that progressed into fibrosis, and not in mild Covid-19. Then, we prospectively followed 83 fILD patients (45% IPF, 55% non-IPF -EAA, CTD-ILD, NSIP-) over 1 year and immunophenotyped them every 3 months. Cross-sectional analysis showed that patients with a higher number circulating MDSC, had a higher GAP index (7-8) (p<0,001). Longitudinal follow-up showed that patients with constant higher circulating MDSC had lower transplant-free survival (p=0.0058). Primary isolated MDSC when co-cultured with autologous T cells induced CD8+ T cell exhaustion (PD1hi, Lag3hi, Tim3hi, TNFalpha lo, INFglo), and downregulation of co-stimulatory T cell signaling (CD28, ICOS, ITK, and LCK), preliminary data support the induction of de-novo FoxP3 Treg formation, creating a suppressive and immunosenescent microenvironment in IPF. FACS analysis of explanted lungs demonstrated the increase of tissue-resident MDSC in fibrosis (HP, NSIP, IPF) compared with donor lungs, as well as in bleomycin-induced fibrosis compared to PBS. Conclusion: Taking together, a high number of circulating MDSC reflects worse lung function and higher GAP index in cross-sectional analysis, and associates with lower transplant-free survival longitudinally. The role that immature and mature monocytes play during promotion of a suppressive microenvironment in IPF is an unexplored area that may lead to a paradigm shift in our understanding of the sequelae of exhaustion and immunosenescence, contributing to the identification of novel targets useful for therapeutic myeloid selection in IPF.

Preserved T-cellresponse in B-cell depleted patientsfollowing SARS-CoV-2 vaccination

Objective: To investigate the humoral and T-cell immune response of multiple sclerosis (MS) patients under B-cell depleting therapy following SARS-CoV-2 vaccination compared to healthy controls (HC). Background: The SARS-CoV-2 pandemic has huge implications for the management of patients with MS under highly active immune therapies. First reports indicated that the humoral response of anti-CD20 treated, B-cell depleted patients might by attenuated. Data about dynamics of B-cell repopulation and T-cell response are scarce. Design/Methods: The study was authorized by the local ethics committee of the RuhrUniversity Bochum. Patients were recruited at the Department of Neurology. We included n=10 healthy age-matched controls and n=37 B-cell depleted MS patients (20 ocrelizumab, 17 off-label rituximab). Serum and Peripheral Blood Mononuclear Cells (PBMCs) were isolated 4-8 weeks after second vaccination. Serum was analyzed for anti-SARS-CoV-2-Spike antibodies. Lymphocyte subpopulations were analyzed in B-cell depleted patients by FACS analysis. PBMCs were stimulated with 2 μg/ml SARS-CoV-2 peptide-mix for 16-18 hours and analyzed via flow cytometry for cytokine-expressing T-cell populations. Results: 15/36 (40.5%) patients mounted an antibody response >10 U/ml (range 0.8-2,500). The anti-SARS-CoV-2-Spike titer correlated with B-cell repopulation (R2 =0.01841;r=0.4481;p=0.0069), whereas no significance could be detected correlating with the time to last infusion (R2 =0.1352;r=0.3058;p=0.0740). On the contrary, T-cell responses of B-cell depleted patients were higher or did not deviate from those of HC. Frequencies of CD4+ -T-cells expressing IFN-γ or IL-4 and CD8+ -T-cells expressing IFN-γ or IFN-γ and IL-2 were significantly higher in B-cell depleted patients, while CD4+ -T-cells expressing IL-2 or IFN-γ and IL-2 as well as CD8+ -T-cells expressing IL-2 did not differ from HC. Conclusions: Humoral vaccination responses in B-cell depleted patients are dependent on B-cell repopulation. Notably, patients with poor humoral response are able to mount a sustained T-cell response after SARS-CoV-2 vaccination. Those data suggest, that vaccinated B-cell depleted patients might be partially protected against SARS-CoV-2 infection.

A RECOMBINANT BIO-HDL (CER-001) CAN PREVENT SARS-COV2-INDUCED RENAL DYSFUNCTION BY RESTORING SR-BI SIGNALLING

BACKGROUND AND AIMS: Replication of the enveloped SARS-COV2 virus can alter lipidomic composition and metabolism of infected cells [1]. These alterations commonly result in a decline in HDL, total cholesterol and LDL, and an increase in triglyceride levels in COVID-19 patients. Furthermore, the 'cytokine storm' subsequent to release of inflammatory cytokines can severely impair lipid homeostasis. Importantly, decreased HDL-cholesterol correlates with severity of COVID-19 infection and represents a significant prognostic factor in predicting poor clinical outcomes [2]. Similarly, it has been observed that COVID-19 patients' recovery is accompanied by a rise in serum HDL levels. Pharmacological intervention that aims to restore ApoA-1 or functional HDL particles may have beneficial roles for clinical outcome of COVID-19 patients and has recently been approved for compassionate use [3]. SARS-CoV 2 spike proteins S1 and S2 can bind free cholesterol and HDL-bound cholesterol, facilitating virus entry by binding the ACE2 co-receptor Scavenger Receptor-BI (SR-BI) [4]. When activated at the trans-membrane level, SR-BI signalling culminates in Ser1173-eNOS phosphorylation with both anti-inflammatory and anti-apoptotic effect. We hypothesized that SARS-COV2 binding promoted SR-BI internalization, so that it could not exert its essential protective function. Therefore, the aim of this study is to evaluate the effects of CER-001, a mimetic HDL, in antagonizing this process. METHOD: Endothelial and tubular (RPTEC) cells were exposed to S1, S2 and S1 + S2 (50-250 nM) with or without CER-001 (CER-001 50-500 ug/mL) and cholesterol (10-50 uM). Apoptosis tests (MTT and AnnV/PI) were performed. Internalization of SR-BI, ACE2 with S1 and activation of eNOS was evaluated by FACS analysis. SR-BI and ACE2 expression were evaluated on kidney biopsies from COVID-19 patients. RESULTS: At concentrations used, the exposition of S1, S2 and S1 + S2 in the presence of CER-001 and cholesterol did not induce apoptosis of endothelial cells and RPTEC. Endothelial and tubular cells stimulated by S1, in presence of cholesterol, showed an increased intracellular level of SR-BI and ACE-2, with significantly reduced eNOS phosphorylation compared to baseline (P < 0.05). The treatment with CER-001 reversed trans-membrane SR-BI levels and eNOS phosphorylation to baseline values. The detection of S1 spike protein by endothelial cells immunohistochemistry revealed an increased level in S1-exposed cells with cholesterol and reduced S1 intracellular positive staining in CER-001-exposed cells (P < 0.05). Interestingly, S1-exposed cells without cholesterol appeared not to be capable of mediating S1 spike protein internalization. Consistent with in vitro results, analysis of renal biopsies from COVID-19 patients with proteinuria showed increased SR-BI and ACE-2 cytoplasmic signals and reduced expression at the apical domain of injured tubules. CONCLUSION: Our data confirmed the key role of lipid profile in SARS-COV2 infection, evaluating the molecular signalling involved in HDL metabolism and inflammatory processes, and could offer new therapeutic strategies for COVID-19 patients. (Figure Presented).

CD64 GENE-MODIFIED PRIMARY NK CELLS LOADED with ANTI-HIV bNAbs TRIGGER HIGH ADCC

Background: As part of a combined HIV CURE immuno-therapy strategy, we transduced primary human NK cells with the high affinity CD64 Fc receptor and pre-loaded them with HIV-specific bNAbs. We named these chimeric NK cells "NuKES" (NK Enhancement Strategy) for their augmented capacity to mediate ADCC and their potential clinical application as an autologous primary NK cell immuno-therapy against HIV. Methods: We transduced primary NK cells from control donors with a lentivirus expressing human CD64 in the presence or absence of irradiated K562 feeder cells expressing co-stimulatory molecules (CD40, 4-1BB) and/or cytokine pre-stimulation (IL-2, IL-21, IL-15). CD64 expressing NK cells were CFSE labeled and expanded ex vivo or FACS sorted at various times post transduction to high purity. CD64 expressing NK cells were then pre-loaded with HIV-specific bNAbs and tested in a functional ADCC CD107a degranulation assay against HIV-1 infected autologous CD4+ primary T cells. Results: After pre-stimulation with cytokines and/or irradiated K562 Feeder Cells, we could routinely achieve (n=5) greater than 40% CD64 expression in primary human NK cells (Day 14 post-transduction shown in Figure 1A). NK cells maintained strong proliferation potential with greater than 6 cells divisions beyond 10 days post transduction as determined by CFSE dilution (Day 10 post-transduction shown in Figure 1B). Phenotypically, CD64 transduced NK cells were similar to control NK cells and possessed strong expression of CD56, CD16, CD69 with intermediate levels of the NK maturation marker CD57. CD64 transduced NK cells could be successfully pre-loaded with HIV-specific bNAbs and possessed an enhanced capacity (GMFI of 2,014 versus 276) to retain 10-1074 for several hours as compared to control NK cells (Figure 1C). Functionally, CD64 transduced NK cells showed a significant two-fold increase in ADCC-triggered degranulation capacity against autologous HIV-1 infected CD4+ primary T cells compared to control NK cells after pre-loading with HIV-specific bNAbs (27.6% versus 13.2% CD107a). Conclusion: Primary human NK cells can be successfully transduced with CD64 and expanded ex vivo to high purity. Preparation of bNAbs specific NuKES represent a viable autologous NK immuno-therapy approach against HIV-1 with potential adaptation for added disease targets (i.e., COVID, Cancer) moving forward.

Pharmacologic inhibition of SUMOactivating enzyme potentiates interferon response and T cell-mediated anti-tumor immunity in chronic lymphocytic leukemia (CLL) and lymphoma models

Introduction: CLL is characterized by deficient immunity which clinically manifests as an increased predisposition toward malignancies and infectious complications. T-cells from patients with CLL exhibit a skewed repertoire with a predominance of Tregs as well as impaired immune synapse formation and cytotoxic function. Unlike chemotherapy, novel targeted agents may have beneficial immunomodulatory effects, which may be particularly relevant in the COVID-19 era. Small ubiquitin-like modifier (SUMO) family proteins regulate a variety of cellular processes, including nuclear trafficking, gene transcription, and cell cycle progression, via post-translational modification of target proteins. Sumoylation regulates NFjB signaling, IFN response, and NFAT activation, processes indispensable in immune cell activation. Despite this, the role of sumoylation in T cell biology in the context of cancer is not known. TAK-981 is a small molecule inhibitor of the SUMO-activating enzyme (SAE) that forms a covalent adduct with an activated SUMO protein, thereby preventing its transfer to the SUMO-conjugating enzyme (Ubc9). Here, we investigated the immunomodulatory effects of TAK-981 in CLL. Methods: T cells from patients with CLL were purified using Dynabeads. Activation, proliferation, and apoptosis of CD3+ T cells were studied following T-cell receptor engagement (TCR;aCD3/CD28) with/without 0-1 lM TAK-981. Cytokines were measured after in vitro stimulation. For polarization assays, FACS-sorted naïve CD4+ T cells were cultured for 7 days in control or differentiation media. For gene expression profiling (GEP;Clariom S), RNA was harvested after 3 and 24 h of TCR engagement from FACS-sorted naïve CD4+ T cells. For in vivo immunization experiments, CD4+KJ1-26+ cells were inoculated IV into BALB/cJ mice. Mice received 100 mg IV ovalbumin ± R848 followed by TAK-981 7.5 mg/kg or vehicle control IV twice weekly for 10 days before spleen collection. Both recipient and transplanted splenocytes were analyzed. For analysis of tumor-infiltrating lymphocytes (TILs), BALB/c mice were injected with 1×106 A20 lymphoma cells and treated as above. TAK-981 was provided by Millennium Pharmaceuticals, Inc. (Cambridge, MA, USA). Results: T cells from patients with CLL demonstrated high baseline protein sumoylation that slightly increased following TCR engagement. Treatment with TAK-981 significantly downregulated SUMO1 and SUMO2/3-modified protein levels, yet did not disrupt early TCR signaling as evidenced by sustained ZAP70, p65/NFjB, and NFAT activation detected by immunoblotting, immunocytochemistry, and GEP. Treatment with TAK-981 resulted in dose-dependent upregulation of the early activation marker CD69 in CD4+ T cells following 72 and 96 h of TCR stimulation vs. control. Meanwhile, the expression of CD25, HLA-DR, and CD40L was delayed in the presence of TAK-981. Interestingly, CD38, an IFN response target, was induced 2-fold in TAK-981-treated cells after 24 h and persisted at high levels at subsequent timepoints. T cell proliferation was reduced in the presence of high (1 lM) but not low/intermediate concentrations of TAK-981, accompanied by reduced S phase entry and decreased synthesis of IL- 2. However, T cells did not undergo apoptosis under those conditions. Targeting SAE in either control or Th1/Treg polarizing conditions facilitated an increase in IFNc and loss of FoxP3 expression (accompanied by decreased IL-2/STAT5), suggesting a shift toward Th1 and away from Treg phenotype, respectively. GEP (Reactome, GSEA) confirmed a dramatically upregulated IFN response in TAK-981-treated CD4+ naïve T cells. Furthermore, targeting SAE enhanced degranulation (CD107a), IFNc, and perforin secretion in cytotoxic CD8+ T cells and potentiated T cell cytotoxicity in allogeneic assays with lymphoma cells (OCI-LY3, U2932) as targets. Consistent with our in vitro data, OVA-stimulated transplanted transgenic KJ1-26+ splenocytes, as well as total CD4+ T cells from recipient mice treated with TAK-981 in vivo exhibited a significant reduction in express on of FoxP3 and an increased production of IFNc. In the A20 syngeneic model, treatment with TAK-981 similarly downregulated FoxP3 expression in CD4+ TILs and induced IFNc secretion in CD8+ TILs. Conclusion: Using a combination of in vitro and in vivo experiments, we demonstrate that pharmacologic targeting of sumoylation with TAK-981 does not impair proximal TCR signaling in T cells obtained from patients with CLL, but leads to rebalancing toward healthy immune T cell subsets via induction of IFN response and downmodulation of Tregs. These data provide a strong rationale for continued investigation of TAK-981 in CLL and lymphoid malignancies.

SARS-CoV-2-specific adaptive immune response after mRNA vaccination in liver transplanted patients

Background and aim Liver transplant recipients have an increased risk to develop severe or prolonged COVID-19. Therefore, these individuals particularly benefit from prophylactic vaccination. Recent studies demonstrated reduced antibody response rates upon COVID-19 mRNA vaccination in immunosuppressed individuals, however, little is known about the role of cellular immunity in this setting. Methods We analyzed T cell responses after bnt162b2 vaccination using overlapping peptides spanning the SARS-CoV-2 spike protein and a set of pre-described epitopes located in different SARS-CoV-2 proteins, as well as the humoral response (serology and neutralizing antibodies) in 10 patients following liver transplantation. Results Importantly, all patients showed T cell and/or antibody responses after vaccination. In line with previous studies, detectable levels of S1-specific IgG and titers of neutralizing antibodies were lower compared to the general vaccinated population. Similarly, the CD4 + and CD8 + T cell epitope repertoire was narrow and cytokine production was reduced. Hypothesizing that the phenotype of T cells is different under immunosuppression with imminent implications for the long-time immunity, we currently perform in-depth phenotypical profiling of the detectable CD8 + T cells using tetramer-based enrichment and multi-parameter FACS analysis. Conclusions Our data suggest an impaired immune response after SARSCoV- 2 vaccination in the vulnerable cohort of individuals after liver transplantation. Cellular immune responses may however compensate for lacking antibody responses. Our data support the notion that immunocompromised patients may benefit from an early third vaccination.

SARS-CoV2 ORF3a protein drives inflammation in COVID-19 through NLRP3 inflammasome activation

Introduction: COVID-19 severe pneumonia has been associated to systemic inflammation and elevation of blood parameters and reminiscent of cytokine storm syndrome. Stimulation of PBMC from patients with severe COVID-19 have shown a high secretion of IL-1β, a pivotal cytokine driving inflammatory phenotypes, which maturation and secretion is regulated by NLRP3 inflammasome. Steroidal anti-inflammatory therapies have shown efficacy in reducing mortality in critically ill patients, however the mechanisms by which SARS-CoV2 virus triggers such an extensive inflammation remain unexplained. Objectives: The overall objective of this study was to investigate if SARS-CoV2 drives inflammation in COVID-19 patients through NLRP3 inflammasome activation and IL-1β secretion. Methods: Samples from SARS-CoV2 infected patients, were collected at day 0 and at 3 and 7 following treatment with anakinra. Fresh monocytes, purified through adherence, were cultured for 3, 6, 18 h in the presence or absence of LPS (100 ng/ml) and MCC950 (10μM). Release of IL-1β, IL-1Ra, IL-6, TNF-α, IL-18 was quantified by ELISA kit. Relative gene expression analysis of ORF3a gene was performed by RT-qPCR. THP-1 cells were transfected with a plasmid containing ORF3a sequence by nucleofection. NLRP3 inflammasome and ASC speck formation were detected by confocal microscopy and/or by FACS analysis. Results: In the present study we show that circulating monocytes from COVID-19 patients display ASC specks, index of NLRP3 activation, and spontaneously secrete IL-1β in vitro. This spontaneous activation reverts following patient's treatment with the IL-1 receptor antagonist anakinra. Transfection of a monocytic cell line with cDNA coding for the ORF3a SARS-CoV2 protein, resulted in NLRP3- dependent ASC speck formation. The involvement of ORF3a in inflammasome activation was further supported by the detection by RT-PCR of ORF3a in monocytes from COVID-19 patients. Conclusion: In summary, these results provide a mechanistic explanation for the strong inflammatory manifestations associated to COVID-19 and further evidence that NLRP3 and IL-1β targeting could represent an effective strategy in this disease.

A Simple Approach for Counting CD4+ T Cells Based on a Combination of Magnetic Activated Cell Sorting and Automated Cell Counting Methods

Frequent tests for CD4+ T cell counting are important for the treatment of patients with immune deficiency;however, the routinely used fluorescence-activated cell-sorting (FACS) gold standard is costly and the equipment is only available in central hospitals. In this study, we developed an alternative simple approach (shortly named as the MACS-Countess system) for CD4+ T cell counting by coupling magnetic activated cell sorting (MACS) to separate CD4+ T cells from blood, followed by counting the separated cells using CountessTM, an automated cell-counting system. Using the cell counting protocol, 25 µL anti-CD4 conjugated magnetic nanoparticles (NP-CD4, BD Bioscience) were optimized for separating CD4+ T cells from 50 µL of blood in PBS using a DynamagTM-2 magnet, followed by the introduction of 10 µL separated cells into a CountessTM chamber slide for automated counting of CD4+ T cells. To evaluate the reliability of the developed method, 48 blood samples with CD4+ T cell concentrations ranging from 105 to 980 cells/µL were analyzed using both MACS-Countess and FACS. Compared with FACS, MACS-Countess had a mean bias of 3.5% with a limit of agreement (LoA) ranging from −36.4% to 43.3%, which is close to the reliability of the commercial product, PIMA analyzer (Alere), reported previously (mean bias 0.2%;LoA ranging from −42% to 42%, FACS as reference). Further, the MACS-Countess system requires very simple instruments, including only a magnet and an automated cell counter, which are affordable for almost every lab located in a limited resource region.