Association of COVID-19 inflammation with activation of the C5a-C5aR1 axis.

Coronavirus disease 2019 (COVID-19) is a disease caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and has resulted in a pandemic1. The C5a complement factor and its receptor C5aR1 (also known as CD88) have a key role in the initiation and maintenance of several inflammatory responses by recruiting and activating neutrophils and monocytes1. Here we provide a longitudinal analysis of immune responses, including phenotypic analyses of immune cells and assessments of the soluble factors that are present in the blood and bronchoalveolar lavage fluid of patients at various stages of COVID-19 severity, including those who were paucisymptomatic or had pneumonia or acute respiratory distress syndrome. The levels of soluble C5a were increased in proportion to the severity of COVID-19 and high expression levels of C5aR1 receptors were found in blood and pulmonary myeloid cells, which supports a role for the C5a-C5aR1 axis in the pathophysiology of acute respiratory distress syndrome. Anti-C5aR1 therapeutic monoclonal antibodies prevented the C5a-mediated recruitment and activation of human myeloid cells, and inhibited acute lung injury in human C5aR1 knock-in mice. These results suggest that blockade of the C5a-C5aR1 axis could be used to limit the infiltration of myeloid cells in damaged organs and prevent the excessive lung inflammation and endothelialitis that are associated with acute respiratory distress syndrome in patients with COVID-19.

Flow cytometric analysis of intracellular phosphoproteins in human monocytes.

BACKGROUND: Using antibodies against intracellular phosphoproteins, flow cytometry can be used to monitor simultaneously multiple signaling pathways. Here, we tested a recently released procedure to analyze phosphorylation events in human monocytes upon different types of stimulation. METHODS: Whole blood was treated by lipopolysaccharide (LPS) or granulocyte-macrophage colony-stimulating factor (GM-CSF), then cells were labeled by antibodies recognizing cell surface and cytosolic proteins. Human monocytes were identified by a CD14 - CD45 staining and three phosphorylated proteins such as AKT, ERK-1/2, and STAT5, were simultaneously detected by multicolor phosphoflow analysis. RESULTS: By this rapid method, we are able to detect directly from a blood sample several signaling events in human monocytes where LPS stimulation induces preferentially ERK-1/2 phosphorylation where as GM-CSF stimulation induces STAT5 phosphorylation. CONCLUSIONS: This procedure provides a simultaneous measurement of multiple activated signaling molecules using a simplified and rapid protocol. © 2015 International Clinical Cytometry Society.

Follicular B Lymphomas Generate Regulatory T Cells via the ICOS/ICOSL Pathway and Are Susceptible to Treatment by Anti-ICOS/ICOSL Therapy.

The prognosis of follicular lymphoma (FL) patients is suspected to be influenced by tumor-infiltrating regulatory T cells (Treg). The mechanism of Treg enrichment in FL and their impact on malignant FL B cells remains to be elucidated. We analyzed 46 fresh lymph node biopsy samples, including FL (n = 20), diffuse large B-cell lymphoma (n = 10), classical Hodgkin lymphoma (n = 9), and reactive lymphadenitis (n = 7). Using multicolor flow cytometry and cell sorting, we observed an accumulation of CD25(high)CD127(low/neg) Tregs in FL tissues. These Tregs comprised activated ICOS(+) Tregs that were able to suppress not only conventional T cells, but also FL B cells. These FL B cells were able to express ICOSL in vitro and to generate CD25(high)FoxP3(high) Tregs expressing ICOS. Treg generation was associated with ICOS/ICOSL engagement and was abrogated by antagonist anti-ICOS and anti-ICOSL antibodies. Interactions between Tregs and FL B cells resulted in ICOSL downregulation on FL B cells. Our results highlight a key role for Tregs in FL pathogenesis and suggest that targeting the ICOS/ICOSL pathway may be a promising immunotherapy for FL treatment. Cancer Res; 76(16); 4648-60. ©2016 AACR.

The co-receptor BTLA negatively regulates human Vγ9Vδ2 T-cell proliferation: a potential way of immune escape for lymphoma cells.

Vγ9Vδ2 cells, the major γδ T-cell subset in human peripheral blood, represent a T-cell subset that displays reactivity against microbial agents and tumors. The biology of Vγ9Vδ2 T cells remains poorly understood. We show herein that the interaction between B- and T-lymphocyte attenuator (BTLA) and herpesvirus entry mediator (HVEM) is a major regulator of Vγ9Vδ2 T-cell proliferation control. BTLA was strongly expressed at the surface of resting Vγ9Vδ2 T cells and inversely correlated with T-cell differentiation. BTLA-HVEM blockade by monoclonal antibodies resulted in the enhancement of Vγ9Vδ2 T-cell receptor-mediated signaling, whereas BTLA-HVEM interaction led to a decrease in phosphoantigen-mediated proliferation by inducing a partial S-phase arrest. Our data also suggested that BTLA-HVEM might participate in the control of γδ T-cell differentiation. In addition, the proliferation of autologous γδ T cells after exposition to lymphoma cells was dramatically reduced through BTLA-HVEM interaction. These data suggest that HVEM interaction with BTLA may play a role in lymphomagenesis by interfering with Vγ9Vδ2 T-cell proliferation. Moreover, BTLA stimulation of Vγ9Vδ2 T cells appears as a new possible mechanism of immune escape by lymphoma cells.

Peripheral blood NK cells from breast cancer patients are tumor-induced composite subsets.

Human NK lymphocytes are involved in antitumor immunity. The therapeutic potential of this population against cancers has stimulated their study and led to the discovery of several NK cell subsets, each of which is endowed with different immunoregulatory functions. We have previously reported that NK cell functions are profoundly altered in advanced breast cancer patients. In this study, we show that these tumor-mediated alterations also variably affect NK cell subsets. We found that in addition to the known human CD56(dim)CD16(+), CD56(bright)CD16(-), and CD56(-)CD16(+) NK cell subsets, two additional subsets, namely the CD56(bright)CD16(+) and CD56(dim)CD16(-) subsets, were increased in the peripheral blood of patients with advanced invasive breast cancers. These subsets corresponded to the main two subsets found at the tumor site. The extensive phenotype of these subsets revealed an "à la carte" pattern of expression for the various NK receptors, functional molecules, adhesion molecules, and chemokine receptors, depending on the subset. We next compared these subsets to known NK cell populations endowed with specific phenotypic characteristics, but also with functional properties. Our data show that advanced breast cancer patients have an increased proportion of more immature and noncytotoxic NK cell subsets in their peripheral blood, which might account for at least part of the low cytotoxic functions observed in these patients. They reveal a major heterogeneity and plasticity of the NK cell compartment, which are both tightly linked to the microenvironment. The identification of NK cell subsets endowed with particular functional capabilities might help monitor residual antitumor NK cell-mediated responses in breast cancer patients.

CpG-ODN-induced sustained expression of BTLA mediating selective inhibition of human B cells.

BTLA (B- and T-lymphocyte attenuator) is a prominent co-receptor that is structurally and functionally related to CTLA-4 and PD-1. In T cells, BTLA inhibits TCR-mediated activation. In B cells, roles and functions of BTLA are still poorly understood and have never been studied in the context of B cells activated by CpG via TLR9. In this study, we evaluated the expression of BTLA depending on activation and differentiation of human B cell subsets in peripheral blood and lymph nodes. Stimulation with CpG upregulated BTLA, but not its ligand: herpes virus entry mediator (HVEM), on B cells in vitro and sustained its expression in vivo in melanoma patients after vaccination. Upon ligation with HVEM, BTLA inhibited CpG-mediated B cell functions (proliferation, cytokine production, and upregulation of co-stimulatory molecules), which was reversed by blocking BTLA/HVEM interactions. Interestingly, chemokine secretion (IL-8 and MIP1ß) was not affected by BTLA/HVEM ligation, suggesting that BTLA-mediated inhibition is selective for some but not all B cell functions. We conclude that BTLA is an important immune checkpoint for B cells, as similarly known for T cells.

PD-1 is a novel regulator of human B-cell activation.

The outcome of the adaptive immune response is determined by the integration of both positive and negative signals, respectively, induced upon the triggering of co-signaling receptors. One of them, programmed cell death 1 (PDCD1/PD-1) has largely been shown to be involved in the negative regulation of T-cell activation. However, PD-1 is also expressed on human B cells, and its role(s) in the process of human B-cell activation remains uncertain thus far. In this study, we describe the expression of PD-1 on the major human B-cells subsets isolated from peripheral blood and lymph nodes. We showed that PD-1 was expressed on naive B cells, was differentially expressed on peripheral IgM memory as compared with memory B cells and was lost on germinal center B cells. Expression of PD-1 ligands (PD-Ls) was induced by TLR9 activation. Finally, we showed that PD-1 was recruited to the B-cell receptor upon triggering. We determined that during TLR9 activation, blockade of PD-1/PD-Ls pathways indeed increased B-cell activation, proliferation and the production of inflammatory cytokines. Altogether, our results show, that, as reported in T cells, PD-1/PD-Ls complexes acted as inhibitors of the B-cell activation cascade and highlight the importance of devising future therapies able to modulate lymphocyte activation through the targeting of the PD-1/PD-Ls pathways.

High expression of indoleamine 2,3-dioxygenase in the tumour is associated with medullary features and favourable outcome in basal-like breast carcinoma.

Medullary breast cancer (MBC) is a basal-like breast carcinoma (BLBC) with a favourable outcome, whereas nonmedullary BLBC has a poor prognosis. Tumour infiltrating lymphocytes (TILs) are present in both MBC and BLBC. We hypothesized that the immunosuppressive enzyme indoleamine 2,3-dioxygenase (IDO) could modulate the TILs effects among these tumours and explain their different outcomes. The amount of TILs and IDO expression were analysed using immunohistochemistry (IHC) in 155 BC cases including MBC (n = 17), atypical MBC (n = 13) and non-MBC (n = 125). Messenger RNA expression of the INDO gene, which encodes IDO, was measured in 262 cases from our institution. INDO mRNA expression and histoclinical data of 1,487 BC cases were collected from public databases. IDO immunostaining was present in both neoplastic and stromal cells in 100% of MBC and was associated with histological medullary features among non-MBC cases. There was a significant correlation between IDO positivity and TIL amounts. In our series including mostly grade-3 BC, IDO immunostaining was the most significant marker (p = 0.02) associated with better survival in multivariate analysis. Among our 262 analysed BC cases, INDO mRNA showed significant overexpression in BLBC as compared to luminal A tumours, and in MBC as compared to basal-like non-MBC. In the pooled series of 1,749 BC cases, INDO mRNA was overexpressed in BLBC and was the most significant predictor of better survival in this subtype using multivariate analysis (p = 0.0024). In conclusion, high IDO expression is associated with morphological medullary features and has an independent favourable prognostic value in BLBC.

Differential role for CD277 as a co-regulator of the immune signal in T and NK cells.

The human butyrophilin (BTN) 3 or CD277 molecules belong to the B7 family members and are expressed in various immune cells such as T and NK cells. Here, we show that CD277 triggering considerably enhances TCR-induced cytokine production and cell proliferation, even when another co-stimulatory molecule, CD28, is engaged. These CD277-induced additive functional effects are in accordance with the detection of early T-cell activation events such as TCR-induced cell signaling being increased upon CD277 engagement. However, we found that CD277 triggering is not involved in CD16- or NKp46-induced NK cell activation. BTN3/CD277 comprises three structurally related members, BTN3A1, BTN3A2 and BTN3A3. CD277 antibodies recognize all isoforms and we describe a differential expression of BTN3 isoforms between T and NK cells that could explain differential CD277 functions between T and NK cells. Our results show that, while T cells express all BTN3/CD277 transcripts, NK cells express mostly BTN3A2, which lacks the B30.2 intracellular domain. Furthermore, NKp30-induced cytokine production is decreased by the specific engagement of BTN3A2, but not by BTN3A1 triggering. Thus, we provide new insights into the CD277 co-stimulatory pathway that may differentially participate in the regulation of various cell-mediated immune responses.

Human breast cancer cells enhance self tolerance by promoting evasion from NK cell antitumor immunity.

NK cells are a major component of the antitumor immune response and are involved in controlling tumor progression and metastases in animal models. Here, we show that dysfunction of these cells accompanies human breast tumor progression. We characterized human peripheral blood NK (p-NK) cells and malignant mammary tumor-infiltrating NK (Ti-NK) cells from patients with noninvasive and invasive breast cancers. NK cells isolated from the peripheral blood of healthy donors and normal breast tissue were used as controls. With disease progression, we found that expression of activating NK cell receptors (such as NKp30, NKG2D, DNAM-1, and CD16) decreased while expression of inhibitory receptors (such as NKG2A) increased and that this correlated with decreased NK cell function, most notably cytotoxicity. Importantly, Ti-NK cells had more pronounced impairment of their cytotoxic potential than p-NK cells. We also identified several stroma-derived factors, including TGF-ß1, involved in tumor-induced reduction of normal NK cell function. Our data therefore show that breast tumor progression involves NK cell dysfunction and that breast tumors model their environment to evade NK cell antitumor immunity. This highlights the importance of developing future therapies able to restore NK cell cytotoxicity to limit/prevent tumor escape from antitumor immunity.

High expression of the inhibitory receptor BTLA in T-follicular helper cells and in B-cell small lymphocytic lymphoma/chronic lymphocytic leukemia.

B- and T-lymphocyte attenuator (BTLA) is a lymphoid receptor that inhibits lymphocyte activation on interaction with its ligand, herpesvirus entry mediator (HVEM). We developed monoclonal antibodies against BTLA and HVEM to study their expression using immunohistochemical and flow cytometric analyses in human tissues. In reactive lymph nodes, they were both expressed in interfollicular T cells and in B cells from mantle and marginal zones. Within germinal centers, B cells were negative, whereas T follicular helper (TFH) cells were BTLA+ and follicular dendritic cells were HVEM+. BTLA was strongly expressed in chronic lymphocytic leukemia/small lymphocytic lymphoma (B-CLL/SLL, 19 of 19 positive) when compared with other small B-cell lymphomas, including follicular lymphoma (0 of 24 positive), mantle cell lymphoma (0 of 10 positive), and marginal zone lymphoma (0 of 5 positive). Our results suggest that down-regulation of the BTLA-HVEM pathway may be involved in germinal center B-cell activation. The specific high expression of BTLA in B-CLL/SLL represents a new potential diagnostic tool. The BTLA positivity of TFH cells may be a basis for designing future immunotherapies.