Role of the PD-1 and PD-L1 axis in COVID-19.

Tweetable abstract Severe COVID-19 patients display dysregulated expression of checkpoint molecules PD-1 and its ligand PD-L1, suggesting that these checkpoint molecules could be considered as prognostic markers and therapeutic targets in severe cases of COVID-19.

Upregulation of PD-1 Expression and High sPD-L1 Levels Associated with COVID-19 Severity.

COVID-19 has several mechanisms that can lead to lymphocyte depletion/exhaustion. The checkpoint inhibitor molecule programmed death protein 1 (PD-1) and its programmed death-ligand 1 (PDL-1) play an important role in inhibiting cellular activity as well as the depletion of these cells. In this study, we evaluated PD-1 expression in TCD4+, TCD8+, and CD19+ lymphocytes from SARS-CoV-2-infected patients. A decreased frequency of total lymphocytes and an increased PD-1 expression in TCD4+ and CD19+ lymphocytes were verified in severe/critical COVID-19 patients. In addition, we found a decreased frequency of total monocytes with an increased PD-1 expression on CD14+ monocytes in severe/critical patients in association with the time of infection. Moreover, we observed an increase in sPD-L1 circulant levels associated with the severity of the disease. Overall, these data indicate an important role of the PD-1/PDL-1 axis in COVID-19 and may provide a severity-associated biomarker and therapeutic target during SARS-CoV-2 infection.

PD-L1 Overexpression in the Lungs of Subjects Who Died from COVID-19: Are We on the Way to Understanding the Immune System Exhaustion Induced by SARS-CoV-2?

Knowledge of the pathogenic mechanisms of severe acute respiratory syndrome-associated coronavirus 2 (SARS-CoV-2) is certainly a priority for the scientific community. Two main elements are involved in the biology of the most severe forms of coronavirus disease 2019 (COVID-19): the direct cytopathic effect of the virus against the host tissues, and a dysfunction of the immune system, characterized by the exhaustion of T lymphocytes. The exhaustion of T cells in COVID-19 is poorly understand, but some data could suggest a possible role of PD-1/PD-L1 axis. The aim of this study was to evaluate the possible role of PD-L1 expression in the pulmonary tissue in subjects affected by COVID-19. The presence of SARS-CoV-2 in the pulmonary tissue, and its exact location, was indagated by in situ hybridization; the expression of PD-L1 and CD8 in the same tissue was indagated by immunohistochemistry. Overall, PD-L1 resulted diffusely expressed in 70% of the cases, and an intense expression was observed in 43.5% of cases. Diffuse and intense presence of SARS-CoV-2 by in situ hybridization significantly correlated with an intense PD-L1 expression, and with expression of PD-L1 by pneumocytes. PD-L1 is overexpressed in the pulmonary tissue of subjects died from COVID-19, and mainly in subjects with a high viral load. These data suggest a possible role of PD-L1 in the immune system exhaustion at the basis of the severe forms of the disease.

Intermediate Monocytes with PD-L1 and CD62L Expression as a Possible Player in Active SARS-CoV-2 Infection.

Monocytes play a role in viral biology, but little is known about the monocyte subpopulation in the course of COVID-19 disease. The aim of the study was the analysis of classical, intermediate and non-classical monocytes with expression of PD-L1 and CD62L, TIM-3 and CD86 molecules in peripheral blood (PB) to distinguish patients with SARS-CoV-2 infection from convalescent patients. The study group consisted of 55 patients with SARS-CoV-2 infection and 51 convalescent patients. The cells were analyzed by flow cytometry. The number and proportion of monocytes were lower in patients with COVID-19 than convalescent patients. We observed a lower proportion of non-classical monocytes in COVID-19 patients than convalescent ones. There was a higher proportion of PDL-1-positive intermediate monocytes in COVID-19 patients than convalescent ones. We noticed a higher geometric mean fluorescence intensity (GeoMean) of PD-L1 on intermediate monocytes in COVID-19 patients than convalescent patients, and a higher proportion of CD62L-positive monocytes in COVID-19 patients in comparison with convalescent ones. We found a higher GeoMean of CD62L on monocytes in COVID-19 patients than convalescent ones. Assessment of PD-L1- and CD62L-positive monocyte subsets may identify patients with a possible predisposition for rapid recovery. The monitoring of monocyte subsets in PB might be a useful test in COVID-19 patients.

SARS-CoV-2 Induces Cytokine Responses in Human Basophils.

Basophils play a key role in the orientation of immune responses. Though the interaction of SARS-CoV-2 with various immune cells has been relatively well studied, the response of basophils to this pandemic virus is not characterized yet. In this study, we report that SARS-CoV-2 induces cytokine responses and in particular IL-13, in both resting and IL-3 primed basophils. The response was prominent under IL-3 primed condition. However, either SARS-CoV-2 or SARS-CoV-2-infected epithelial cells did not alter the expression of surface markers associated with the activation of basophils, such as CD69, CD13 and/or degranulation marker CD107a. We also validate that human basophils are not permissive to SARS-CoV-2 replication. Though increased expression of immune checkpoint molecule PD-L1 has been reported on the basophils from COVID-19 patients, we observed that SARS-CoV-2 does not induce PD-L1 on the basophils. Our data suggest that basophil cytokine responses to SARS-CoV-2 might help in reducing the inflammation and also to promote antibody responses to the virus.

Vitamin D can reduce severity in COVID-19 through regulation of PD-L1.

COVID-19 is a highly contagious viral infection that has killed millions of people around the world. The most important diagnostic feature of COVID-19 is lymphocyte depletion, particularly the depletion of T cells. In COVID-19 infections, there is a link between destruction of T cells and increased expression of inhibitory immune checkpoint molecules (PD-1/PD-L1) on T cell surfaces. It was shown that PD-1/PD-L1 levels increase in severely COVID-19 infected individuals. Higher proinflammatory cytokine levels cause increased PD-1/PD-L1 expression. In severe COVID-19, higher proinflammatory cytokine levels may increase PD-1/PD-L1. Vitamin-D is an important immune regulator. It is known that the numbers of CD4+ and CD8+ T lymphocytes decrease in vitamin D deficiency while vitamin D supplementation increases CD + 4 lymphocytes. Vitamin D can increase regulatory T cell (Treg) activity. Vitamin D also has a diminishing effect on proinflammatory cytokines. In severe COVID-19 cases, vitamin D supplementation may inhibit the increase of PD-L1 expression through reducing proinflammatory cytokine levels. Thus, vitamin D supplementation could eliminate the suppressive effect of PD-L1 on CD4+ and CD8+ T cells, preventing lymphopenia and reducing disease severity and mortality in patients infected with COVID-19. Besides, vitamin D supplementation can reduce inflammation by increasing Treg activity. The aim of this letter is to discuss the functions of inhibitory immune checkpoint molecules and their effects on dysfunction and depletion of T-cells as well as to explain the possible modulatory effect of vitamin D on these checkpoints and T cells.

Platelets modulate CD4+ T-cell function in COVID-19 through a PD-L1 dependent mechanism.

Severe COVID-19 is associated with a systemic inflammatory response and progressive CD4+ T-cell lymphopenia and dysfunction. We evaluated whether platelets might contribute to CD4+ T-cell dysfunction in COVID-19. We observed a high frequency of CD4+ T cell-platelet aggregates in COVID-19 inpatients that inversely correlated with lymphocyte counts. Platelets from COVID-19 inpatients but not from healthy donors (HD) inhibited the upregulation of CD25 expression and tumour necrosis factor (TNF)-α production by CD4+ T cells. In addition, interferon (IFN)-γ production was increased by platelets from HD but not from COVID-19 inpatients. A high expression of PD-L1 was found in platelets from COVID-19 patients to be inversely correlated with IFN-γ production by activated CD4+ T cells cocultured with platelets. We also found that a PD-L1-blocking antibody significantly restored platelets' ability to stimulate IFN-γ production by CD4+ T cells. Our study suggests that platelets might contribute to disease progression in COVID-19 not only by promoting thrombotic and inflammatory events, but also by affecting CD4+ T cells functionality.

Unbiased Analysis of Temporal Changes in Immune Serum Markers in Acute COVID-19 Infection With Emphasis on Organ Failure, Anti-Viral Treatment, and Demographic Characteristics.

Identification of novel immune biomarkers to gauge the underlying pathology and severity of COVID-19 has been difficult due to the lack of longitudinal studies. Here, we analyzed serum collected upon COVID-19 admission (t1), 48 hours (t2), and seven days later (t3) using Olink proteomics and correlated to clinical, demographics, and therapeutic data. Older age positively correlated with decorin, pleiotrophin, and TNFRS21 but inversely correlated with chemokine (both C-C and C-X-C type) ligands, monocyte attractant proteins (MCP) and TNFRS14. The burden of pre-existing conditions was positively correlated with MCP-4, CAIX, TWEAK, TNFRS12A, and PD-L2 levels. Individuals with COVID-19 demonstrated increased expression of several chemokines, most notably from the C-C and C-X-C family, as well as MCP-1 and MCP-3 early in the course of the disease. Similarly, deceased individuals had elevated MCP-1 and MCP-3 as well as Gal-9 serum levels. LAMP3, GZMB, and LAG3 at admission correlated with mortality. Only CX3CL13 and MCP-4 correlated positively with APACHE score and length of stay, while decorin, MUC-16 and TNFRSF21 with being admitted to the ICU. We also identified several organ-failure-specific immunological markers, including those for respiratory (IL-18, IL-15, Gal-9) or kidney failure (CD28, VEGF). Treatment with hydroxychloroquine, remdesivir, convalescent plasma, and steroids had a very limited effect on the serum variation of biomarkers. Our study identified several potential targets related to COVID-19 heterogeneity (MCP-1, MCP-3, MCP-4, TNFR superfamily members, and programmed death-ligand), suggesting a potential role of these molecules in the pathology of COVID-19.

PD-L1 Dysregulation in COVID-19 Patients.

The COVID-19 pandemic has reached direct and indirect medical and social consequences with a subset of patients who rapidly worsen and die from severe-critical manifestations. As a result, there is still an urgent need to identify prognostic biomarkers and effective therapeutic approaches. Severe-critical manifestations of COVID-19 are caused by a dysregulated immune response. Immune checkpoint molecules such as Programmed death-1 (PD-1) and its ligand programmed death-ligand 1 (PD-L1) play an important role in regulating the host immune response and several lines of evidence underly the role of PD-1 modulation in COVID-19. Here, by analyzing blood sample collection from both hospitalized COVID-19 patients and healthy donors, as well as levels of PD-L1 RNA expression in a variety of model systems of SARS-CoV-2, including in vitro tissue cultures, ex-vivo infections of primary epithelial cells and biological samples obtained from tissue biopsies and blood sample collection of COVID-19 and healthy individuals, we demonstrate that serum levels of PD-L1 have a prognostic role in COVID-19 patients and that PD-L1 dysregulation is associated to COVID-19 pathogenesis. Specifically, PD-L1 upregulation is induced by SARS-CoV-2 in infected epithelial cells and is dysregulated in several types of immune cells of COVID-19 patients including monocytes, neutrophils, gamma delta T cells and CD4+ T cells. These results have clinical significance since highlighted the potential role of PD-1/PD-L1 axis in COVID-19, suggest a prognostic role of PD-L1 and provide a further rationale to implement novel clinical studies in COVID-19 patients with PD-1/PD-L1 inhibitors.

Concentrated Secretome of Adipose Stromal Cells Limits Influenza A Virus-Induced Lung Injury in Mice.

Despite vaccination and antivirals, influenza remains a communicable disease of high burden, with limited therapeutic options available to patients that develop complications. Here, we report the development and preclinical characterization of Adipose Stromal Cell (ASC) concentrated secretome (CS), generated by process adaptable to current Good Manufacturing Practices (cGMP) standards. We demonstrate that ASC-CS limits pulmonary histopathological changes, infiltration of inflammatory cells, protein leak, water accumulation, and arterial oxygen saturation (spO2) reduction in murine model of lung infection with influenza A virus (IAV) when first administered six days post-infection. The ability to limit lung injury is sustained in ASC-CS preparations stored at -80 °C for three years. Priming of the ASC with inflammatory factors TNFα and IFNγ enhances ASC-CS ability to suppress lung injury. IAV infection is associated with dramatic increases in programmed cell death ligand (PDL1) and angiopoietin 2 (Angpt2) levels. ASC-CS application significantly reduces both PDL1 and Angpt2 levels. Neutralization of PDL1 with anti-mouse PDL1 antibody starting Day6 onward effectively ablates lung PDL1, but only non-significantly reduces Angpt2 release. Most importantly, late-phase PDL1 neutralization results in negligible suppression of protein leakage and inflammatory cell infiltration, suggesting that suppression of PDL1 does not play a critical role in ASC-CS therapeutic effects.

The pathogenic role of epithelial and endothelial cells in early-phase COVID-19 pneumonia: victims and partners in crime.

Current understanding of the complex pathogenesis of COVID-19 interstitial pneumonia pathogenesis in the light of biopsies carried out in early/moderate phase and histology data obtained at postmortem analysis is discussed. In autopsies the most observed pattern is diffuse alveolar damage with alveolar-epithelial type-II cell hyperplasia, hyaline membranes, and frequent thromboembolic disease. However, these observations cannot explain some clinical, radiological and physiopathological features observed in SARS-CoV-2 interstitial pneumonia, including the occurrence of vascular enlargement on CT and preserved lung compliance in subjects even presenting with or developing respiratory failure. Histological investigation on early-phase pneumonia on perioperative samples and lung biopsies revealed peculiar morphological and morpho-phenotypical changes including hyper-expression of phosphorylated STAT3 and immune checkpoint molecules (PD-L1 and IDO) in alveolar-epithelial and endothelial cells. These features might explain in part these discrepancies.

Comments on the ambiguity of selected surface markers, signaling pathways and omics profiles hampering the identification of myeloid-derived suppressor cells.

Myeloid-derived suppressor cells (MDSC) are important immune-regulatory cells but their identification remains difficult. Here, we provide a critical view on selected surface markers, transcriptional and translational pathways commonly used to identify MDSC by specific, their developmental origin and new possibilities by transcriptional or proteomic profiling. Discrimination of MDSC from their non-suppressive counterparts is a prerequisite for the development of successful therapies. Understanding the switch mechanisms that direct granulocytic and monocytic development into a pro-inflammatory or anti-inflammatory direction will be crucial for therapeutic strategies. Manipulation of these myeloid checkpoints are exploited by tumors and pathogens, such as M. tuberculosis (Mtb), HIV or SARS-CoV-2, that induce MDSC for immune evasion. Thus, specific markers for MDSC identification may reveal also novel molecular candidates for therapeutic intervention at the level of MDSC.

SARS-CoV-2 infection: The role of PD-1/PD-L1 and CTLA-4 axis.

The outbreak of SARS-CoV-2 in Wuhan of China in December 2019 and its worldwide spread has turned into the COVID-19 pandemic. Respiratory disorders, lymphopenia, cytokine cascades, and the immune responses provoked by this virus play a major and fundamental role in the severity of the symptoms and the immunogenicity which it causes. Owing to the decrease in the inflammatory responses' regulation in the immune system and the sudden increase in the secretion of cytokines, it seems that an investigation of inhibitory immune checkpoints can influence theories regarding this disease's treatment methods. Acquired cell-mediated immune defense's T-cells have a key major contribution in clearing viral infections thus reducing the severity of COVID-19's symptoms. The most important diagnostic feature in individuals with COVID-19 is lymphocyte depletion, most importantly, T-cells. Due to the induction of interferon-γ (INF-γ) production by neutrophils and monocytes, which are abundantly present in the peripheral blood of the individuals with COVID-19, the expression of inhibitory immune checkpoints including, PD-1 (programmed death), PD-L1 and CTLA4 on the T-cells' surface is enhanced. The purpose of this review is to discuss the functions of these checkpoints and their effects on the dysfunction and exhaustion of T-cells, making them almost ineffective in individuals with COVID-19, especially in the cases with extreme symptoms.

At the Crossroads: COVID-19 and Immune-Checkpoint Blockade for Cancer.

The immunomodulatory effects of immune-checkpoint blockade (ICB) therapy for cancer may act at the crossroads between the need to increase antiviral immune responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and to decrease the inflammatory responses in severe cases of coronavirus disease 2019 (COVID-19). There is evidence from preclinical models that blocking programmed death receptor 1 (PD1) protects against RNA virus infections, which suggests that patients with cancer receiving ICB may have lower rates of viral infection. However, given the heterogeneity of patient characteristics, this would be difficult to demonstrate using population-based registries or in clinical trials. Most studies of the impact of ICB therapy on the course of COVID-19 have centered on studying its potential detrimental impact on the course of the COVID-19 infection, in particular on the development of the most severe inflammatory complications. This is a logical concern as it is becoming clear that complications of COVID-19 such as severe respiratory distress syndrome are related to interferon signaling, which is the pathway that leads to expression of the PD1 ligand PD-L1. Therefore, PD1/PD-L1 ICB could potentially increase inflammatory processes, worsening the disease course for patients. However, review of the current evidence does not support the notion that ICB therapy worsens complications from COVID-19, and we conclude that it supports the continued use of ICB therapy during the COVID-19 pandemic provided that we now collect data on the effects of such therapy on COVID-19 vaccination.

Efficacy of chemotherapy combined with toripalimab in PD-L1-positive and high tumor mutation burden pancreatic acinar cell carcinoma: case report.

BACKGROUND: Pancreatic acinar cell carcinoma (PACC) is a rare tumor, accounting for about 1% of all pancreatic exocrine cancers. Consensus on the management of metastatic PACC remains unclear. CASE PRESENTATION: Starting from April 2019, a patient first received chemotherapy with two cycles of gemcitabine and nab-paclitaxel and two cycles of SOX regimen. After progression of disease evaluated based on RECIST 1.1, toripalimab and SOX regimen was administered because of PD-L1-positive expression, high tumor mutation burden (TMB), and somatic FANCA deletion in the tumor. Both the primary and metastatic tumor mass shrank significantly after two courses. The patient exhibited sustained partial response for at least six courses with well-controlled toxic effects. Then the treatment had to be stopped for 2 months because of the coronavirus disease 2019 pandemic. Computed tomography scan in March 2020 showed disease progression. Time from initiating treatment to tumor progression on toripalimab and SOX regimen treatment took up to at least 8 months. CONCLUSIONS: We present the first case report where a PD-L1 positive, high TMB, and FANCA-deleted pancreatic acinar cell carcinoma was treated using chemotherapy combined with immunotherapy, in which the patient exhibited satisfactory response and tolerance.

Disharmonic Inflammatory Signatures in COVID-19: Augmented Neutrophils' but Impaired Monocytes' and Dendritic Cells' Responsiveness.

COVID-19, caused by SARS-CoV-2 virus, emerged as a pandemic disease posing a severe threat to global health. To date, sporadic studies have demonstrated that innate immune mechanisms, specifically neutrophilia, NETosis, and neutrophil-associated cytokine responses, are involved in COVID-19 pathogenesis; however, our understanding of the exact nature of this aspect of host-pathogen interaction is limited. Here, we present a detailed dissection of the features and functional profiles of neutrophils, dendritic cells, and monocytes in COVID-19. We portray the crucial role of neutrophils as drivers of hyperinflammation associated with COVID-19 disease via the shift towards their immature forms, enhanced degranulation, cytokine production, and augmented interferon responses. We demonstrate the impaired functionality of COVID-19 dendritic cells and monocytes, particularly their low expression of maturation markers, increased PD-L1 levels, and their inability to upregulate phenotype upon stimulation. In summary, our work highlights important data that prompt further research, as therapeutic targeting of neutrophils and their associated products may hold the potential to reduce the severity of COVID-19.