ABSTRACT
Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been associated with immune hyperactivation and high levels of proinflammatory cytokines. Extensive lung infiltration by CD169+ inflammatory monocytes and presence of activated CD169+ alveolar macrophages suggest monocyte/macrophages are key drivers of severe morbidity and mortality. In this study, we determined whether CD169 mediated ACE2-independent SARS-CoV-2 entry and restricted viral genome replication in macrophages triggers pro-inflammatory cytokine expression. Methods: Monocyte-derived macrophages (MDMs) and PMA-differentiated THP-1 macrophages engineered to constitutively express CD169, ACE2, or CD169 and ACE2 were infected with USA-WA1/2020/SARS-CoV-2 isolate with or without Remdesivir pre-treatment. To identify mechanism of innate immune activation, nucleic acid sensing pathways were selectively depleted in CD169+ macrophages. Extent of viral genomic (gRNA) and sub-genomic (sgRNA) expression and induction of pro-inflammatory cytokines was determined by qRT-PCR and single molecule RNA FISH analysis. Viral protein expression and infectious virus particle production was determined by immunofluorescence analysis and TCID50. Results: While productive virus infection (viral protein expression and infectious virus particle release) was only observed in ACE2+ macrophages, SARS-CoV-2 N or S expression and infectious virus production was not observed in CD169+ macrophages. Co-expression of ACE2 and CD169 significantly enhanced infectious virus production and spread. Interestingly, smFISH and RT-qPCR analysis revealed CD169+ cells express cytosolic negative-strand gRNA and positive strand sgRNA. Importantly, CD169-mediated SARS-CoV-2 infection of macrophages and expression of viral mRNAs led to induction of pro-inflammatory cytokines, IL-6, TNFα, and IL-1β, despite lack of viral protein expression in CD169+ macrophages. Pre-treatment with Remdesivir blocked de novo expression of viral mRNAs and induction of inflammatory cytokines in CD169-dependent infection of macrophages. Furthermore, knockdown of cytosolic RLRs (RIG-I and MDA-5) or MAVS significantly attenuated inflammatory cytokine expression in CD169+ macrophages, confirming that nucleic acid sensing of restricted cytosolic viral mRNA expression in macrophages triggers innate immune activation. Conclusion: These results suggest that restricted SARS-CoV-2 infection of CD169+ macrophages contributes to COVID-19-associated hyperinflammatory cytokine response.
ABSTRACT
INTRODUCTION We have previously described AUTO1, a CD19 CAR with a fast off-rate CD19 binding domain, designed to reduce CAR-T immune toxicity and improve engraftment. Its clinical activity has been tested in r/r paediatric and adult B-ALL. Cumulatively, this data confirms the intended function of the receptor, with low levels of CRS/ICANS and long-term engraftment of CAR T-cells observed in both patient groups. Recently, CAR-T therapy has been explored in indolent lymphomas such as follicular (FL) and mantle cell lymphoma (MCL), but a high incidence of toxicity including Grade 3-4 ICANS has been reported. We have initiated testing of AUTO1 in the setting of indolent and high-grade B-NHL and CLL (NCT02935257). METHODS Manufacturing: CAR T-cell products were generated using a semi-automated closed process from non-mobilised leukapheresate. Study design: Subjects ≥ 16y underwent lymphodepletion with fludarabine (30mg/m 2 x3) and cyclophosphamide (60mg/kg x1) prior to AUTO1 infusion, with the exception of the DLBCL cohort who additionally received a single dose of pembrolizumab (200mg) on day -1 to potentiate CAR-T expansion. AUTO1 dose varies based on the indication. Split dosing of 230 x10
ABSTRACT
Background: We have previously described AUTO1 (CAT19), a CD19 CAR with a fast off-rate CD19 binding domain, designed to reduce CAR T-cell immune toxicity and improve engraftment. Its clinical activity has been tested in r/r paediatric and adult B-ALL. Cumulatively, this data confirms the intended fucntion of the receptor, with low levels of CRS/ ICANS and long-term engraftment of CAR T-cells observed in both patient groups. Recently, CAR therapy has been explored in indolent lymphomas such as follicluar (FL) and mantle cell lymphoma (MCL), but a high incidence of toxicity inluding Grade 3-4 ICANS has been reported. Aims: We have initiated testing of AUTO1 (CAT19) in the setting of indolent B-cell lymphoma (NCT02935257). Methods: Manufacturing: CAR T-cell products were generated using a semi-automated closed process from non-mobilised leucapheresate. Study design: subjects>/=16y underwent lymhpodepletion with fludarabine (30mg/m2 x3) and cyclophosphamide (60mg/kg x1) followed by a single CAR T-cell infusion of 200 x10