Dendritic cells can prime anti-tumor CD8+ T cell responses through major histocompatibility complex cross-dressing.

Antigen cross-presentation, wherein dendritic cells (DCs) present exogenous antigen on major histocompatibility class I (MHC-I) molecules, is considered the primary mechanism by which DCs initiate tumor-specific CD8+ T cell responses. Here, we demonstrate that MHC-I cross-dressing, an antigen presentation pathway in which DCs acquire and display intact tumor-derived peptide:MHC-I molecules, is also important in orchestrating anti-tumor immunity. Cancer cell MHC-I expression was required for optimal CD8+ T cell activation in two subcutaneous tumor models. In vivo acquisition of tumor-derived peptide:MHC-I molecules by DCs was sufficient to induce antigen-specific CD8+ T cell priming. Transfer of tumor-derived human leukocyte antigen (HLA) molecules to myeloid cells was detected in vitro and in human tumor xenografts. In conclusion, MHC-I cross-dressing is crucial for anti-tumor CD8+ T cell priming by DCs. In addition to quantitatively enhancing tumor antigen presentation, MHC cross-dressing might also enable DCs to more faithfully and efficiently mirror the cancer cell peptidome.

Wild-Type U2AF1 Antagonizes the Splicing Program Characteristic of U2AF1-Mutant Tumors and Is Required for Cell Survival.

We have asked how the common S34F mutation in the splicing factor U2AF1 regulates alternative splicing in lung cancer, and why wild-type U2AF1 is retained in cancers with this mutation. A human lung epithelial cell line was genetically modified so that U2AF1S34F is expressed from one of the two endogenous U2AF1 loci. By altering levels of mutant or wild-type U2AF1 in this cell line and by analyzing published data on human lung adenocarcinomas, we show that S34F-associated changes in alternative splicing are proportional to the ratio of S34F:wild-type gene products and not to absolute levels of either the mutant or wild-type factor. Preferential recognition of specific 3' splice sites in S34F-expressing cells is largely explained by differential in vitro RNA-binding affinities of mutant versus wild-type U2AF1 for those same 3' splice sites. Finally, we show that lung adenocarcinoma cell lines bearing U2AF1 mutations do not require the mutant protein for growth in vitro or in vivo. In contrast, wild-type U2AF1 is required for survival, regardless of whether cells carry the U2AF1S34F allele. Our results provide mechanistic explanations of the magnitude of splicing changes observed in U2AF1-mutant cells and why tumors harboring U2AF1 mutations always retain an expressed copy of the wild-type allele.

T-cell States, Repertoire, and Function in Classical Hodgkin Lymphoma Revealed through Single-Cell Analyses.

The classical Hodgkin lymphoma (cHL) environment is comprised of a dense and complex immune cell infiltrate interspersed with rare malignant Hodgkin-Reed-Sternberg (HRS) cells. HRS cells are actively surveilled by endogenous T cells, but data linking phenotypic and functional T-cell states with clonality at the single-cell level in cHL is lacking. To address this knowledge gap, we performed paired single-cell RNA and T-cell receptor sequencing on 14 cHL and 5 reactive lymphoid tissue specimens. Conventional CD4+ T cells dominated the cHL landscape. However, recurrent clonal expansion within effector and exhausted CD8+ T-cell and regulatory T-cell clusters was uniquely observed in cHL specimens. Multiplex flow cytometric analysis revealed that most lymphoma-resident T cells produced effector cytokines upon ex vivo restimulation, arguing against a profound dysfunctional T-cell state in cHL. Our results raise new questions about the nature of T cells that mediate the antilymphoma response following programmed cell death protein 1 (PD-1) blockade therapy in cHL.

CD5 Gene Signature Identifies Diffuse Large B-Cell Lymphomas Sensitive to Bruton's Tyrosine Kinase Inhibition.

PURPOSE: A genetic classifier termed LymphGen accurately identifies diffuse large B-cell lymphoma (DLBCL) subtypes vulnerable to Bruton's tyrosine kinase inhibitors (BTKis), but is challenging to implement in the clinic and fails to capture all DLBCLs that benefit from BTKi-based therapy. Here, we developed a novel CD5 gene expression signature as a biomarker of response to BTKi-based therapy in DLBCL. METHODS: CD5 immunohistochemistry (IHC) was performed on 404 DLBCLs to identify CD5 IHC+ and CD5 IHC- cases, which were subsequently characterized at the molecular level through mutational and transcriptional analyses. A 60-gene CD5 gene expression signature (CD5sig) was constructed using genes differentially expressed between CD5 IHC+ and CD5 IHC- non-germinal center B-cell-like (non-GCB DLBCL) DLBCLs. This CD5sig was applied to external DLBCL data sets, including pretreatment biopsies from patients enrolled in the PHOENIX study (n = 584) to define the extent to which the CD5sig could identify non-GCB DLBCLs that benefited from the addition of ibrutinib to rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). RESULTS: CD5 expression was observed in 12% of non-GCB DLBCLs. CD5+ DLBCLs displayed transcriptional features of B-cell receptor (BCR) activation and were enriched for BCR-activating mutations known to correlate with BTKi sensitivity. However, most CD5+ DLBCLs lacked canonical BCR-activating mutations or were LymphGen-unclassifiable (LymphGen-Other). The CD5sig recapitulated these findings in multiple independent data sets, indicating its utility in identifying DLBCLs with genetic and nongenetic bases for BCR dependence. Supporting this notion, CD5sig+ DLBCLs derived a selective survival advantage from the addition of ibrutinib to R-CHOP in the PHOENIX study, independent of LymphGen classification. CONCLUSION: CD5sig is a useful biomarker to identify DLBCLs vulnerable to BTKi-based therapies and complements current biomarker approaches by identifying DLBCLs with genetic and nongenetic bases for BTKi sensitivity.

Integrative genomic analysis identifies unique immune environments associated with immunotherapy response in diffuse large B cell lymphoma.

Most diffuse large B-cell lymphoma (DLBCL) patients treated with bispecific antibodies (BsAb) or chimeric antigen receptor (CAR) T cells fail to achieve durable treatment responses, underscoring the need for a deeper understanding of mechanisms that regulate the immune environment and response to treatment. Here, an integrative, multi-omic approach was employed to characterize DLBCL immune environments, which effectively segregated DLBCLs into four quadrants - termed DLBCL-immune quadrants (IQ) - defined by cell-of-origin and immune-related gene set expression scores. Recurrent genomic alterations were enriched in each IQ, suggesting that lymphoma cell-intrinsic alterations contribute to orchestrating unique DLBCL immune environments. In relapsed/refractory DLBCL patients, DLBCL-IQ assignment correlated significantly with clinical benefit with the CD20 x CD3 BsAb, mosunetuzumab, but not with CD19-directed CAR T cells. DLBCL-IQ provides a new framework to conceptualize the DLBCL immune landscape and uncovers the differential impact of the endogenous immune environment on outcomes to BsAb and CAR T cell treatment.

Pembrolizumab for the treatment of disease relapse after allogeneic hematopoietic stem cell transplantation.

A failed graft-versus-tumor (GVT) effect is a common mechanism of relapse after allogeneic hematopoietic cell transplantation (alloHCT). Although targeting the PD-1/PD-L1 axis may restore GVT effects, PD-1 blockade exacerbates graft-versus-host disease (GVHD) in murine models, and severe GVHD can occur in patients treated with anti-PD-1 therapy after alloHCT. Therefore, we developed a prospective study to assess the safety and efficacy of pembrolizumab in patients relapsing after alloHCT. Eligible patients received pembrolizumab (200 mg every 3 weeks) for up to 2 years. Twelve patients were enrolled (8 patients with acute myeloid leukemia, 1 patient with myelodysplastic syndrome, 1 patient with classical Hodgkin lymphoma, and 2 patients with diffuse large B-cell lymphoma [DLBCL]). All participants received reduced-intensity preparative regimens with in vivo T-cell depletion. The median time from alloHCT to enrollment was 587 days (range, 101-4211). Three participants (25%) experienced grade 3 to 4 immune-related adverse events (irAE) (pneumonitis, 2 patients; hyperthyroidism, 1 patient), all occurring after 1 to 2 cycles, and resolving after pembrolizumab discontinuation and corticosteroid treatment. irAEs of any grade occurred in 5 patients (42%). No treatment-emergent GVHD was observed. Overall and complete response (CR) rates were 22% (2/9). Both patients achieving CRs had PD-L1 gene-amplified lymphomas and diffuse PD-L1 expression on pretreatment biopsies. An acquired EZH2 mutation was identified at relapse in a patient with DLBCL who achieved an initial CR to pembrolizumab, which was associated with downregulated HLA expression on malignant B cells, implicating EZH2 mutations as a potential immune escape mechanism after PD-1-blockade therapy. In conclusion, after alloHCT, treatment with pembrolizumab is feasible and associated with objective responses in relapsed lymphoid malignancies but can induce severe irAEs, requiring vigilant monitoring. This trial was registered at www.clinicaltrials.gov as #NCT02981914.

TIGIT is a key inhibitory checkpoint receptor in lymphoma.

BACKGROUND: PD-1 checkpoint blockade therapy (CBT) has greatly benefited patients with select solid tumors and lymphomas but has limited efficacy against diffuse large B-cell lymphoma (DLBCL). Because numerous inhibitory checkpoint receptors have been implicated in driving tumor-specific T cell dysfunction, we hypothesized that combinatorial CBT would enhance the activity of anti-PD-1-based therapy in DLBCL. T cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT) is a coinhibitory receptor expressed on dysfunctional tumor-infiltrating T cells, and TIGIT blockade has demonstrated encouraging activity in combination with PD-1 blockade in murine tumor models and in clinical studies. However, the degree to which TIGIT mediates T cell dysfunction in DLBCL has not been fully explored. RESULTS: Here, we demonstrate that TIGIT is broadly expressed on lymphoma-infiltrating T cells (LITs) across a variety of human lymphomas and is frequently coexpressed with PD-1. TIGIT expression is particularly common on LITs in DLBCL, where TIGIT+ LITs often form distinct cellular communities and exhibit significant contact with malignant B cells. TIGIT+/PD-1+ LITs from human DLBCL and murine lymphomas exhibit hypofunctional cytokine production on ex vivo restimulation. In mice with established, syngeneic A20 B-cell lymphomas, TIGIT or PD-1 mono-blockade leads to modest delays in tumor outgrowth, whereas PD-1 and TIGIT co-blockade results in complete rejection of A20 lymphomas in most mice and significantly prolongs survival compared with mice treated with monoblockade therapy. CONCLUSIONS: These results provide rationale for clinical investigation of TIGIT and PD-1 blockade in lymphomas, including DLBCL.

Efficacy of checkpoint inhibition after CAR-T failure in aggressive B-cell lymphomas: outcomes from 15 US institutions.

Checkpoint inhibitor (CPI) therapy with anti-PD-1 antibodies has been associated with mixed outcomes in small cohorts of patients with relapsed aggressive B-cell lymphomas after CAR-T failure. To define CPI therapy efficacy more definitively in this population, we retrospectively evaluated clinical outcomes in a large cohort of 96 patients with aggressive B-cell lymphomas receiving CPI therapy after CAR-T failure across 15 US academic centers. Most patients (53%) had diffuse large B-cell lymphoma, were treated with axicabtagene ciloleucel (53%), relapsed early (≤180 days) after CAR-T (83%), and received pembrolizumab (49%) or nivolumab (43%). CPI therapy was associated with an overall response rate of 19% and a complete response rate of 10%. Median duration of response was 221 days. Median progression-free survival (PFS) and overall survival (OS) were 54 and 159 days, respectively. Outcomes to CPI therapy were significantly improved in patients with primary mediastinal B-cell lymphoma. PFS (128 vs 51 days) and OS (387 vs 131 days) were significantly longer in patients with late (>180 days) vs early (≤180 days) relapse after CAR-T. Grade ≥3 adverse events occurred in 19% of patients treated with CPI. Most patients (83%) died, commonly because of progressive disease. Only 5% had durable responses to CPI therapy. In the largest cohort of patients with aggressive B-cell lymphoma treated with CPI therapy after CAR-T relapse, our results reveal poor outcomes, particularly among those relapsing early after CAR-T. In conclusion, CPI therapy is not an effective salvage strategy for most patients after CAR-T, where alternative approaches are needed to improve post-CAR-T outcomes.

Checkpoint Blockade-Induced Dermatitis and Colitis Are Dominated by Tissue-Resident Memory T Cells and Th1/Tc1 Cytokines.

Immune checkpoint blockade is therapeutically successful for many patients across multiple cancer types. However, immune-related adverse events (irAE) frequently occur and can sometimes be life threatening. It is critical to understand the immunologic mechanisms of irAEs with the goal of finding novel treatment targets. Herein, we report our analysis of tissues from patients with irAE dermatitis using multiparameter immunofluorescence (IF), spatial transcriptomics, and RNA in situ hybridization (RISH). Skin psoriasis cases were studied as a comparison, as a known Th17-driven disease, and colitis was investigated as a comparison. IF analysis revealed that CD4+ and CD8+ tissue-resident memory T (TRM) cells were preferentially expanded in the inflamed portion of skin in cutaneous irAEs compared with healthy skin controls. Spatial transcriptomics allowed us to focus on areas containing TRM cells to discern functional phenotype and revealed expression of Th1-associated genes in irAEs, compared with Th17-asociated genes in psoriasis. Expression of PD-1, CTLA-4, LAG-3, and other inhibitory receptors was observed in irAE cases. RISH technology combined with IF confirmed expression of IFNγ, CXCL9, CXCL10, and TNFα in irAE dermatitis, as well as IFNγ within TRM cells specifically. The Th1-skewed phenotype was confirmed in irAE colitis cases compared with healthy colon.

Antibody and T cell responses to COVID-19 vaccination in patients receiving anticancer therapies.

BACKGROUND: Patients with cancer were excluded from phase 3 COVID-19 vaccine trials, and the immunogenicity and side effect profiles of these vaccines in this population is not well understood. Patients with cancer can be immunocompromised from chemotherapy, corticosteroids, or the cancer itself, which may affect cellular and/or humoral responses to vaccination. PD-1 is expressed on T effector cells, T follicular helper cells and B cells, leading us to hypothesize that anti-PD-1 immunotherapies may augment antibody or T cell generation after vaccination. METHODS: Antibodies to the SARS-CoV-2 receptor binding domain (RBD) and spike protein were assessed in patients with cancer (n=118) and healthy donors (HD, n=22) after 1, 2 or 3 mRNA vaccine doses. CD4+ and CD8+ T cell reactivity to wild-type (WT) or B.1.617.2 (delta) spike peptides was measured by intracellular cytokine staining. RESULTS: Oncology patients without prior COVID-19 infections receiving immunotherapy (n=36), chemotherapy (n=15), chemoimmunotherapy (n=6), endocrine or targeted therapies (n=6) and those not on active treatment (n=26) had similar RBD and Spike IgG antibody titers to HDs after two vaccinations. Contrary to our hypothesis, PD-1 blockade did not augment antibody titers or T cell responses. Patients receiving B-cell directed therapies (n=14) including anti-CD20 antibodies and multiple myeloma therapies had decreased antibody titers, and 9/14 of these patients were seronegative for RBD antibodies. No differences were observed in WT spike-reactive CD4+ and CD8+ T cell generation between treatment groups. 11/13 evaluable patients seronegative for RBD had a detectable WT spike-reactive CD4+ T cell response. T cells cross-reactive against the B.1.617.2 variant spike peptides were detected in 31/59 participants. Two patients with prior immune checkpoint inhibitor-related adrenal insufficiency had symptomatic hypoadrenalism after vaccination. CONCLUSIONS: COVID-19 vaccinations are safe and immunogenic in patients with solid tumors, who developed similar antibody and T cell responses compared with HDs. Patients on B-cell directed therapies may fail to generate RBD antibodies after vaccination and should be considered for prophylactic antibody treatments. Many seronegative patients do develop a T cell response, which may have an anti-viral effect. Patients with pre-existing adrenal insufficiency may need to take stress dose steroids during vaccination to avoid adrenal crisis.

Severe COVID-19 induces autoantibodies against angiotensin II that correlate with blood pressure dysregulation and disease severity.

Patients infected with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can experience life-threatening respiratory distress, blood pressure dysregulation, and thrombosis. This is thought to be associated with an impaired activity of angiotensin-converting enzyme 2 (ACE2), which is the main entry receptor of SARS-CoV-2 and which also tightly regulates blood pressure by converting the vasoconstrictive peptide angiotensin II (AngII) to a vasopressor peptide. Here, we show that a significant proportion of hospitalized patients with COVID-19 developed autoantibodies against AngII, whose presence correlates with lower blood oxygenation, blood pressure dysregulation, and overall higher disease severity. Anti-AngII antibodies can develop upon specific immune reaction to the SARS-CoV-2 proteins Spike or receptor-binding domain (RBD), to which they can cross-bind, suggesting some epitope mimicry between AngII and Spike/RBD. These results provide important insights on how an immune reaction against SARS-CoV-2 can impair blood pressure regulation.

Quantitative tissue analysis and role of myeloid cells in non-small cell lung cancer.

BACKGROUND: Despite the prominent role of innate immunity in the antitumor response, little is known about the myeloid composition of human non-small cell lung cancer (NSCLC) with respect to histology and molecular subtype. We used multiplexed quantitative immunofluorescence (QIF) to measure the distribution and clinical significance of major myeloid cell subsets in large retrospective NSCLC collections. METHODS: We established a QIF panel to map major myeloid cell subsets in fixed human NSCLC including 4',6-Diamidino-2-Phenylindole for all cells, pancytokeratin for tumor-epithelial cells, CD68 for M1-like macrophages; and CD11b plus HLA-DR to interrogate mature and immature myeloid cell populations such as myeloid derived suppressor cells (MDSCs). We interrogated 793 NSCLCs represented in four tissue microarray-based cohorts: #1 (Yale, n=379) and #2 (Greece, n=230) with diverse NSCLC subtypes; #3 (Yale, n=138) with molecularly annotated lung adenocarcinomas (ADC); and #4 (Yale, n=46) with patient-matched NSCLC and morphologically-normal lung tissue. We examined associations between marker levels, myeloid cell profiles, clinicopathologic/molecular variables and survival. RESULTS: The levels of CD68+ M1 like macrophages were significantly lower and the fraction of CD11b+/HLA-DR- MDSC-like cells was prominently higher in tumor than in matched non-tumor lung tissues. HLA-DR was consistently higher in myeloid cells from tumors with elevated CD68 expression. Stromal CD11b was significantly higher in squamous cell carcinomas (SCC) than in ADC across the cohorts and EGFR-mutated lung ADCs displayed lower CD11b levels than KRAS-mutant tumors. Increased stromal CD68- and HLA-DR-expressing cells was associated with better survival in ADCs from two independent NSCLC cohorts. In SCC, increased stromal CD11b or HLA-DR expression was associated with a trend towards shorter 5-year survival. CONCLUSIONS: NSCLCs display an unfavorable myeloid immune contexture relative to non-tumor lung and exhibit distinct myeloid-cell profiles across histologies and presence of major oncogenic driver-mutations. Elevated M1-like stromal proinflammatory myeloid cells are prognostic in lung ADC, but not in SCC.

Lymphatic coagulation and neutrophil extracellular traps in lung-draining lymph nodes of COVID-19 decedents.

Clinical manifestations of severe COVID-19 include coagulopathies that are exacerbated by the formation of neutrophil extracellular traps (NETs). Here, we report that pulmonary lymphatic vessels, which traffic neutrophils and other immune cells to the lung-draining lymph node (LDLN), can also be blocked by fibrin clots in severe COVID-19. Immunostained tissue sections from COVID-19 decedents revealed widespread lymphatic clotting not only in the lung but also in the LDLN, where the extent of clotting correlated with the presence of abnormal, regressed, or missing germinal centers (GCs). It strongly correlated with the presence of intralymphatic NETs. In mice, tumor necrosis factor α induced intralymphatic fibrin clots; this could be inhibited by DNase I, which degrades NETs. In vitro, TNF-α induced lymphatic endothelial cell upregulation of ICAM-1 and CXCL8, among other neutrophil-recruiting factors, as well as thrombomodulin downregulation; in decedents, lymphatic clotting in LDLNs. In a separate cohort of hospitalized patients, serum levels of Myeloperoxidase-DNA (MPO-DNA, a NET marker) inversely correlated with antiviral antibody titers, but D-dimer levels, indicative of blood thrombosis, did not correlate with either. Patients with high MPO-DNA but low D-dimer levels generated poor antiviral antibody titers. This study introduces lymphatic coagulation in lungs and LDLNs as a clinical manifestation of severe COVID-19 and suggests the involvement of NETosis of lymphatic-trafficking neutrophils. It further suggests that lymphatic clotting may correlate with impaired formation or maintenance of GCs necessary for robust antiviral antibody responses, although further studies are needed to determine whether and how lymphatic coagulation affects adaptive immune responses.

Immune cell and tumor cell-derived CXCL10 is indicative of immunotherapy response in metastatic melanoma.

A T cell-inflamed tumor microenvironment is characterized by the accumulation and local activation of CD8+ T cells and Bat3-lineage dendritic cells, which together are associated with clinical response to anti-programmed cell death protein 1 (anti-PD-1)-based immunotherapy. Preclinical models have demonstrated a crucial role for the chemokine CXCL10 in the recruitment of effector CD8+ T cells into the tumor site, and a chemokine gene signature is also seen in T cell-inflamed tumors from patients. However, the cellular source of CXCL10 in human solid tumors is not known. To identify the cellular source of CXCL10 we analyzed 22 pretreatment biopsy samples of melanoma metastases from patients who subsequently underwent checkpoint blockade immunotherapy. We stained for CD45+ and Sox10+ cells with multiparameter immunofluorescence staining, and RNA in situ hybridization technology was used in concert to identify CXCL10 transcripts. The results were correlated with the expression levels of CXCL10 transcripts from bulk RNA sequencing and the best overall response to immune checkpoint inhibition (anti-PD-1 alone or with anti-CTLA-4) in the same patients. We identified CD45+ cells as the major cellular source for CXCL10 in human melanoma metastases, with additional CXCL10 production seen by Sox10+ cells. Up to 90% of CD45+ cells and up to 69% of Sox10+ cells produced CXCL10 transcripts. The CXCL10 staining result was consistent with the level of CXCL10 expression determined by bulk RNA sequencing. The percentages of CD45+ CXCL10+ cells and Sox10+ CXCL10+ cells independently predicted response (p<0.001). The average number of transcripts per cell correlated with the CD45+ cell infiltrate (R=0.37). Immune cells and melanoma cells produce CXCL10 in human melanoma metastases. Intratumoral CXCL10 is a positive prognostic factor for response to immunotherapy, and the RNAscope technique is achievable using paraffin tissue. Strategies that support effector T cell recruitment via induction of CXCL10 should be considered as a mechanism-based intervention to expand immunotherapy efficacy.

Brachytherapy and non-cancer mortality in patients with oral cavity and oropharynx SCCs.

BACKGROUND: Oral cavity and oropharyngeal squamous cell cancers (OC-OPSCC) display high cancer-specific mortality and increased non-cancer mortality. We examined cause of death in patients treated for OC-OPSCC with brachytherapy, chemotherapy, external beam radiation, surgery, or combination of modalities. We hypothesized that brachytherapy does not increase non-cancer mortality comparably with external beam radiation. METHODS: A database was constructed from institutional tumor registry and electronic medical record data from all patients with first OC-OPSCC diagnosis at our institution between 2000 and 2010, excluding patients with a second primary cancer at diagnosis. The primary outcome was association between treatment modality and non-cancer mortality. RESULTS: Of 693 eligible patients, 460 were deceased; 84 from primary malignancy and 96 from a non-primary cancer cause, including 24 with a second primary cancer. 193 patients received brachytherapy. Cox proportional hazards regression was performed on treatment regimen, stratified by AJCC stage, race, and sex. Age, smoking history, and alcohol had HRs for death of 1.05 (p < 0.005), 1.37 (p = 0.106), and 2.24 (p < 0.005), respectively, while brachytherapy had a 0.53 HR (p < 0.005) for death. Non-smoking OPC patients had an 88% 5-year OS, suggesting these were largely HPV-driven cancers. In smoking OPC patients, 5-year OS was 61%. Non-cancer mortality HR of 0.36 for brachytherapy-treated patients. CONCLUSION: We report non-cancer mortality from a cohort of curatively treated OC-OPSCC and show a significant correlation between brachytherapy and non-cancer survival, independent of remission status. The impact of brachytherapy in OPC was strongest in smokers.

SARS-CoV-2 infection induces cross-reactive autoantibodies against angiotensin II.

Patients infected with the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) can experience life-threatening respiratory distress, blood pressure dysregulation and thrombosis. This is thought to be associated with an impaired activity of angiotensin-converting enzyme-2 (ACE-2), which is the main entry receptor of SARS-CoV-2 and which also tightly regulates blood pressure by converting the vasoconstrictive peptide angiotensin II (AngII) to a vasopressor peptide. Here, we show that a significant proportion of hospitalized COVID-19 patients developed autoantibodies against AngII, whose presence correlates with lower blood oxygenation, blood pressure dysregulation, and overall higher disease severity. Anti-AngII antibodies can develop upon specific immune reaction to the SARS-CoV-2 proteins Spike or RBD, to which they can cross-bind, suggesting some epitope mimicry between AngII and Spike/RBD. These results provide important insights on how an immune reaction against SARS-CoV-2 can impair blood pressure regulation.

Severe COVID-19 infection is associated with aberrant cytokine production by infected lung epithelial cells rather than by systemic immune dysfunction.

The mechanisms explaining progression to severe COVID-19 remain poorly understood. It has been proposed that immune system dysregulation/over-stimulation may be implicated, but it is not clear how such processes would lead to respiratory failure. We performed comprehensive multiparameter immune monitoring in a tightly controlled cohort of 128 COVID-19 patients, and used the ratio of oxygen saturation to fraction of inspired oxygen (SpO2 / FiO2) as a physiologic measure of disease severity. Machine learning algorithms integrating 139 parameters identified IL-6 and CCL2 as two factors predictive of severe disease, consistent with the therapeutic benefit observed with anti-IL6-R antibody treatment. However, transcripts encoding these cytokines were not detected among circulating immune cells. Rather, in situ analysis of lung specimens using RNAscope and immunofluorescent staining revealed that elevated IL-6 and CCL2 were dominantly produced by infected lung type II pneumocytes. Severe disease was not associated with higher viral load, deficient antibody responses, or dysfunctional T cell responses. These results refine our understanding of severe COVID-19 pathophysiology, indicating that aberrant cytokine production by infected lung epithelial cells is a major driver of immunopathology. We propose that these factors cause local immune regulation towards the benefit of the virus.

COVIDOSE: A Phase II Clinical Trial of Low-Dose Tocilizumab in the Treatment of Noncritical COVID-19 Pneumonia.

Interleukin-6 (IL-6)-mediated hyperinflammation may contribute to the mortality of coronavirus disease 2019 (COVID-19). The IL-6 receptor-blocking monoclonal antibody tocilizumab has been repurposed for COVID-19, but prospective trials and dose-finding studies in COVID-19 have not yet fully reported. We conducted a single-arm phase II trial of low-dose tocilizumab in nonintubated hospitalized adult patients with COVID-19, radiographic pulmonary infiltrate, fever, and C-reactive protein (CRP) ≥ 40 mg/L. We hypothesized that doses significantly lower than the emerging standards of 400 mg or 8 mg/kg would resolve clinical and laboratory indicators of hyperinflammation. A dose range from 40 to 200 mg was evaluated, with allowance for one repeat dose at 24 to 48 hours. The primary objective was to assess the relationship of dose to fever resolution and CRP response. Thirty-two patients received low-dose tocilizumab, with the majority experiencing fever resolution (75%) and CRP decline consistent with IL-6 pathway abrogation (86%) in the 24-48 hours following drug administration. There was no evidence of a relationship between dose and fever resolution or CRP decline over the dose range of 40-200 mg. Within the 28-day follow-up, 5 (16%) patients died. For patients who recovered, median time to clinical recovery was 3 days (interquartile range, 2-5). Clinically presumed and/or cultured bacterial superinfections were reported in 5 (16%) patients. Low-dose tocilizumab was associated with rapid improvement in clinical and laboratory measures of hyperinflammation in hospitalized patients with COVID-19. Results of this trial provide rationale for a randomized, controlled trial of low-dose tocilizumab in COVID-19.