High-dimensional mass cytometry analysis of NK cell alterations in AML identifies a subgroup with adverse clinical outcome.

Natural killer (NK) cells are major antileukemic immune effectors. Leukemic blasts have a negative impact on NK cell function and promote the emergence of phenotypically and functionally impaired NK cells. In the current work, we highlight an accumulation of CD56-CD16+ unconventional NK cells in acute myeloid leukemia (AML), an aberrant subset initially described as being elevated in patients chronically infected with HIV-1. Deep phenotyping of NK cells was performed using peripheral blood from patients with newly diagnosed AML (n = 48, HEMATOBIO cohort, NCT02320656) and healthy subjects (n = 18) by mass cytometry. We showed evidence of a moderate to drastic accumulation of CD56-CD16+ unconventional NK cells in 27% of patients. These NK cells displayed decreased expression of NKG2A as well as the triggering receptors NKp30 and NKp46, in line with previous observations in HIV-infected patients. High-dimensional characterization of these NK cells highlighted a decreased expression of three additional major triggering receptors required for NK cell activation, NKG2D, DNAM-1, and CD96. A high proportion of CD56-CD16+ NK cells at diagnosis was associated with an adverse clinical outcome and decreased overall survival (HR = 0.13; P = 0.0002) and event-free survival (HR = 0.33; P = 0.018) and retained statistical significance in multivariate analysis. Pseudotime analysis of the NK cell compartment highlighted a disruption of the maturation process, with a bifurcation from conventional NK cells toward CD56-CD16+ NK cells. Overall, our data suggest that the accumulation of CD56-CD16+ NK cells may be the consequence of immune escape from innate immunity during AML progression.

Dok1 and Dok2 proteins regulate natural killer cell development and function.

Natural killer (NK) cells are involved in immune responses against tumors and microbes. NK-cell activation is regulated by intrinsic and extrinsic mechanisms that ensure NK tolerance and efficacy. Here, we show that the cytoplasmic signaling molecules Dok1 and Dok2 are tyrosine phosphorylated upon NK-cell activation. Overexpression of Dok proteins in human NK cells reduces cell activation induced by NK-cell-activating receptors. Dok1 and Dok2 gene ablation in mice induces an NK-cell maturation defect and leads to increased IFN-γ production induced by activating receptors. Taken together, these results reveal that Dok1 and Dok2 proteins are involved in an intrinsic negative feedback loop downstream of NK-cell-activating receptors in mouse and human.

Fit αß T-cell receptor suppresses leukemogenesis of Pten-deficient thymocytes.

Signaling through the αßT cell receptor (TCR) is a crucial determinant of T-cell fate and can induce two opposite outcomes during thymocyte development: cell death or survival and differentiation. To date, the role played by T-cell receptor in the oncogenic transformation of developing T cells remains unclear. Here we show that human primary T-cell acute lymphoblastic leukemias expressing an αßT cell receptor are frequently deficient for phosphatase and tensin homolog protein (PTEN), and fail to respond strongly to T-cell receptor activation. Using Pten-deficient T-cell acute lymphoblastic leukemia mouse models, we confirm that T-cell receptor signaling is involved in leukemogenesis. We show that abrogation of T-cell receptor expression accelerated tumor onset, while enforced expression of a fit transgenic T-cell receptor led to the development of T-cell receptor-negative lymphoma and delayed tumorigenesis. We further demonstrate that pre-tumoral Pten-deficient thymocytes harboring fit T-cell receptors undergo early clonal deletion, thus preventing their malignant transformation, while cells with unfit T-cell receptors that should normally be deleted during positive selection, pass selection and develop T-cell acute lymphoblastic leukemias. Altogether, our data show that fit T-cell receptor signaling suppresses tumor development mediated by Pten loss-of-function and point towards a role of Pten in positive selection.

Dok1 and Dok2 Proteins Regulate Cell Cycle in Hematopoietic Stem and Progenitor Cells.

Dok1 and Dok2 proteins play a crucial role in myeloid cell proliferation as demonstrated by Dok1 and Dok2 gene inactivation, which induces a myeloproliferative disease in aging mice. In this study, we show that Dok1/Dok2 deficiency affects myeloproliferation even at a young age. An increase in the cellularity of multipotent progenitors is observed in young Dok1/Dok2-deficient mice. This is associated with an increase in the cells undergoing cell cycle, which is restricted to myeloid committed progenitors. Furthermore, cellular stress triggered by 5-fluorouracil (5-FU) treatment potentiates the effects of the loss of Dok proteins on multipotent progenitor cell cycle. In addition, Dok1/Dok2 deficiency induces resistance to 5-FU-induced hematopoietic stem cell exhaustion. Taken together, these results demonstrate that Dok1 and Dok2 proteins are involved in the control of hematopoietic stem cell cycle regulation.

In the absence of its cytosolic domain, the CD28 molecule still contributes to T cell activation.

The CD28 costimulatory receptor has a pivotal role in T cell biology as this molecule amplifies T cell receptor (TCR) signals to provide an efficient immune T cell response. There is a large debate about how CD28 mediates these signals. Here, we designed a CD28 gene-targeted knock-in mouse strain lacking the cytoplasmic tail of CD28. As is the case in CD28-deficient (CD28 knock-out) mice, regulatory T cell homeostasis and T cell activation are altered in these CD28 knock-in mice. Unexpectedly, the presence of a CD28 molecule deprived of its cytoplasmic tail could partially induce some early activation events in T cells such as signaling events or expression of early activation markers. These results unravel a new mechanism of T cell costimulation by CD28, independent of its cytoplasmic tail.

Control of CD8 T cell proliferation and terminal differentiation by active STAT5 and CDKN2A/CDKN2B.

CD8 T cells used in adoptive immunotherapy may be manipulated to optimize their effector functions, tissue-migratory properties and long-term replicative potential. We reported that antigen-stimulated CD8 T cells transduced to express an active form of the transcription factor signal transducer and activator of transcription 5 (STAT5CA) maintained these properties upon adoptive transfer. We now report on the requirements of STAT5CA-expressing CD8 T cells for cell survival and proliferation in vivo. We show that STAT5CA expression allows for greater expansion of T cells in vivo, while preserving dependency on T-cell-receptor-mediated tonic stimulation for their in vivo maintenance and return to a quiescent stage. STAT5CA expression promotes the formation of a large pool of effector memory T cells that respond upon re-exposure to antigen and present an increased sensitivity to γc receptor cytokine engagement for STAT5 phosphorylation. In addition, STAT5CA expression prolongs the survival of what would otherwise be short-lived terminally differentiated KLRG1-positive effector cells with up-regulated expression of the senescence-associated p16(INK) (4A) transcripts. However, development of a KLRG1-positive CD8 T cell population was independent of either p16(INK) (4A) or p19(ARF) expression (as shown using T cells from CDKN2A(-/-) mice) but was associated with expression of transcripts encoding p15(INK) (4B) , another protein involved in senescence induction. We conclude that T-cell-receptor- and cytokine-dependent regulation of effector T cell homeostasis, as well as mechanisms leading to senescent features of a population of CD8 T cells are maintained in STAT5CA-expressing CD8 T cells, even for cells that are genetically deficient in expression of the tumour suppressors p16(INK) (4A) and p19(ARF) .

HCV glycoprotein E2 is a novel BDCA-2 ligand and acts as an inhibitor of IFN production by plasmacytoid dendritic cells.

The elimination of hepatitis C virus (HCV) in > 50% of chronically infected patients by treatment with IFN-α suggests that plasmacytoid dendritic cells (pDCs), major producers of IFN-α, play an important role in the control of HCV infection. However, despite large amounts of Toll-like receptor 7-mediated IFN-α, produced by pDCs exposed to HCV-infected hepatocytes, HCV still replicates in infected liver. Here we show that HCV envelope glycoprotein E2 is a novel ligand of pDC C-type lectin immunoreceptors (CLRs), blood DC antigen 2 (BDCA-2) and DC-immunoreceptor (DCIR). HCV particles inhibit, via binding of E2 glycoprotein to CLRs, production of IFN-α and IFN-λ in pDCs exposed to HCV-infected hepatocytes, and induce in pDCs a rapid phosphorylation of Akt and Erk1/2, in a manner similar to the crosslinking of BDCA-2 or DCIR. Blocking of BDCA-2 and DCIR with Fab fragments of monoclonal antibodies preserves the capacity of pDCs to produce type I and III IFNs in the presence of HCV particles. Thus, negative interference of CLR signaling triggered by cell-free HCV particles with Toll-like receptor signaling triggered by cell-associated HCV results in the inhibition of the principal pDC function, production of IFN.

Constitutive AKT activation in follicular lymphoma.

BACKGROUND: The phosphoinositide 3- kinase (PI3K) pathway is involved in the growth of various human cancers, including lymphoid malignancies. However its role in the pathogenesis of follicular lymphoma (FL) has not been yet described. METHODS: To clarify this point, biopsy tissue samples from 38 human FL cases were investigated for PIK3CA somatic mutations in exon 9 and 20 using direct sequencing. The same samples were analyzed using western blotting and immunohistochemistry to detect expression of AKT, phosphorylated AKT (pAKT), and PTEN proteins. Two cases of benign lymphadenitis were used as controls. RESULTS: AKT expression was present in all FL and lymphadenitis cases. 14/38 (37%) FL and 2/2 lymphadenitis cases expressed pAKT. 9/38 (24%) FL samples showed high level of pAKT, whereas 5/38 (13%) FL cases and 2/2 benign lymphadenitis samples expressed low level of pAKT. PTEN expression was observed in 30/38 (79%) FL and 2/2 benign lymphadenitis cases, whereas 8/38 (21%) FL cases showed loss of PTEN expression. 3 cases with positive pAKT did not express PTEN. PIK3CA mutations were not detected in any sample. CONCLUSIONS: These data suggest that the PI3K/AKT signaling pathway could be activated in a subset of FL cases, due to either AKT phosphorylation or PTEN downregulation, in the absence of PIK3CA mutations.

DOK1 and DOK2 regulate CD8 T cell signaling and memory formation without affecting tumor cell killing.

Targeting intracellular inhibiting proteins has been revealed to be a promising strategy to improve CD8+ T cell anti-tumor efficacy. Here, we are focusing on intracellular inhibiting proteins specific to TCR signaling: DOK1 and DOK2 expressed in T cells. We hypothesized that depletion of intracellular inhibition checkpoint DOK1 and DOK2 could improve CD8+ T-cell based cancer therapies. To evaluate the role of DOK1 and DOK2 depletion in physiology and effector function of CD8+ T lymphocytes and in cancer progression, we established a transgenic T cell receptor mouse model specific to melanoma antigen hgp100 (pmel-1 TCR Tg) in WT and Dok1/Dok2 DKO (double KO) mice. We showed that both DOK1 and DOK2 depletion in CD8+ T cells after an in vitro pre-stimulation induced a higher percentage of effector memory T cells as well as an up regulation of TCR signaling cascade- induced by CD3 mAbs, including the increased levels of pAKT and pERK, two major phosphoproteins involved in T cell functions. Interestingly, this improved TCR signaling was not observed in naïve CD8+ T cells. Despite this enhanced TCR signaling essentially shown upon stimulation via CD3 mAbs, pre-stimulated Dok1/Dok2 DKO CD8+ T cells did not show any increase in their activation or cytotoxic capacities against melanoma cell line expressing hgp100 in vitro. Altogether we demonstrate here a novel aspect of the negative regulation by DOK1 and DOK2 proteins in CD8+ T cells. Indeed, our results allow us to conclude that DOK1 and DOK2 have an inhibitory role following long term T cell stimulations.

Hepatitis C virus fails to activate NF-κB signaling in plasmacytoid dendritic cells.

Plasmacytoid dendritic cells (pDCs) respond to viral infection by production of alpha interferon (IFN-α), proinflammatory cytokines, and cell differentiation. The elimination of hepatitis C virus (HCV) in more than 50% of chronically infected patients by treatment with IFN-α suggests that pDCs can play an important role in the control of HCV infection. pDCs exposed to HCV-infected hepatoma cells, in contrast to cell-free HCV virions, produce large amounts of IFN-α. To further investigate the molecular mechanism of HCV sensing, we studied whether exposure of pDCs to HCV-infected hepatoma cells activates, in parallel to interferon regulatory factor 7 (IRF7)-mediated production of IFN-α, nuclear factor kappa B (NF-κB)-dependent pDC responses, such as expression of the differentiation markers CD40, CCR7, CD86, and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and secretion of the proinflammatory cytokines TNF-α and interleukin 6 (IL-6). We demonstrate that exposure of pDCs to HCV-infected hepatoma cells surprisingly did not induce phosphorylation of NF-κB or cell surface expression of CD40, CCR7, CD86, or TRAIL or secretion of TNF-α and IL-6. In contrast, CpG-A and CpG-B induced production of TNF-α and IL-6 in pDCs exposed to the HCV-infected hepatoma cells, showing that cell-associated virus did not actively inhibit Toll-like receptor (TLR)-mediated NF-κB phosphorylation. Our results suggest that cell-associated HCV signals in pDCs via an endocytosis-dependent mechanism and IRF7 but not via the NF-κB pathway. In spite of IFN-α induction, cell-associated HCV does not induce a full functional response of pDCs. These findings contribute to the understanding of evasion of immune responses by HCV.

Negative intracellular regulators of T-cell receptor (TCR) signaling as potential antitumor immunotherapy targets.

Immunotherapy strategies aim to mobilize immune defenses against tumor cells by targeting mainly T cells. Co-inhibitory receptors or immune checkpoints (ICPs) (such as PD-1 and CTLA4) can limit T cell receptor (TCR) signal propagation in T cells. Antibody-based blocking of immune checkpoints (immune checkpoint inhibitors, ICIs) enable escape from ICP inhibition of TCR signaling. ICI therapies have significantly impacted the prognosis and survival of patients with cancer. However, many patients remain refractory to these treatments. Thus, alternative approaches for cancer immunotherapy are needed. In addition to membrane-associated inhibitory molecules, a growing number of intracellular molecules may also serve to downregulate signaling cascades triggered by TCR engagement. These molecules are known as intracellular immune checkpoints (iICPs). Blocking the expression or the activity of these intracellular negative signaling molecules is a novel field of action to boost T cell-mediated antitumor responses. This area is rapidly expanding. Indeed, more than 30 different potential iICPs have been identified. Over the past 5 years, several phase I/II clinical trials targeting iICPs in T cells have been registered. In this study, we summarize recent preclinical and clinical data demonstrating that immunotherapies targeting T cell iICPs can mediate regression of solid tumors including (membrane associated) immune-checkpoint inhibitor refractory cancers. Finally, we discuss how these iICPs are targeted and controlled. Thereby, iICP inhibition is a promising strategy opening new avenues for future cancer immunotherapy treatments.

Anti-HVEM mAb therapy improves antitumoral immunity both in vitro and in vivo, in a novel transgenic mouse model expressing human HVEM and BTLA molecules challenged with HVEM expressing tumors.

BACKGROUND: Tumor necrosis factor superfamily member 14 (TNFRSF14)/herpes virus entry mediator (HVEM) is the ligand for B and T lymphocyte attenuator (BTLA) and CD160-negative immune co-signaling molecules as well as viral proteins. Its expression is dysregulated with an overexpression in tumors and a connection with tumors of adverse prognosis. METHODS: We developed C57BL/6 mouse models co-expressing human (hu)BTLA and huHVEM as well as antagonistic monoclonal antibodies (mAbs) that completely prevent the interactions of HVEM with its ligands. RESULTS: Here, we show that the anti-HVEM18-10 mAb increases primary human αß-T cells activity alone (CIS-activity) or in the presence of HVEM-expressing lung or colorectal cancer cells in vitro (TRANS-activity). Anti-HVEM18-10 synergizes with antiprogrammed death-ligand 1 (anti-PD-L1) mAb to activate T cells in the presence of PD-L1-positive tumors, but is sufficient to trigger T cell activation in the presence of PD-L1-negative cells. In order to better understand HVEM18-10 effects in vivo and especially disentangle its CIS and TRANS effects, we developed a knockin (KI) mouse model expressing human BTLA (huBTLA+/+) and a KI mouse model expressing both huBTLA+/+/huHVEM+/+ (double KI (DKI)). In vivo preclinical experiments performed in both mouse models showed that HVEM18-10 treatment was efficient to decrease human HVEM+ tumor growth. In the DKI model, anti-HVEM18-10 treatment induces a decrease of exhausted CD8+ T cells and regulatory T cells and an increase of effector memory CD4+ T cells within the tumor. Interestingly, mice which completely rejected tumors (±20%) did not develop tumors on rechallenge in both settings, therefore showing a marked T cell-memory phenotype effect. CONCLUSIONS: Altogether, our preclinical models validate anti-HVEM18-10 as a promising therapeutic antibody to use in clinics as a monotherapy or in combination with existing immunotherapies (antiprogrammed cell death protein 1/anti-PD-L1/anti-cytotoxic T-lymphocyte antigen-4 (CTLA-4)).

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.

A pharmacological strategy to recapitulate exercise-induced antitumoral immunity.

The antitumor activity of exercise by means of enhanced immune activation is documented, but better identification of the underlying mechanisms is required to develop new therapeutic strategies. Recent work from the Dr Bar-Sagi group reveals that exercise engages IL-15 signaling and pharmacological activation of the IL-15/IL-15R axis mimics the exercise-driven immune cell-mediated cytotoxicity in pancreatic cancer.

CISH Expression Is Associated with Metastasis-Free Interval in Triple-Negative Breast Cancer and Refines the Prognostic Value of PDL1 Expression.

Strategies are being explored to increase the efficiency of immune checkpoint inhibitors (ICIs) targeting PD1/PDL1 in triple-negative breast cancer (TNBC), including combination with therapies inhibiting intracellular immune checkpoints such as CISH (Cytokine-induced SH2 protein). Correlation between CISH expression and TNBC features is unknown. We retrospectively analyzed CISH expression in 1936 clinical TNBC samples and searched for correlations with clinical variables, including metastasis-free interval (MFI). Among TNBCs, 44% were identified as "CISH-up" and 56% "CISH-down". High expression was associated with pathological axillary lymph node involvement, more adjuvant chemotherapy, and Lehmann's immunomodulatory and luminal AR subtypes. The "CISH-up" class showed longer 5-year MFI (72%) than the "CISH-down" class (60%; p = 2.8 × 10-2). CISH upregulation was associated with activation of IFNα and IFNγ pathways, antitumor cytotoxic immune response, and signatures predictive for ICI response. When CISH and PDL1 were upregulated together, the 5-year MFI was 81% versus 52% when not upregulated (p = 6.21 × 10-6). The two-gene model provided more prognostic information than each gene alone and maintained its prognostic value in multivariate analysis. CISH expression is associated with longer MFI in TNBC and refines the prognostic value of PDL1 expression. Such observation might reinforce the therapeutic relevance of combining CISH inhibition with an anti-PD1/PDL1 ICI.

High-throughput mass cytometry staining for deep phenotyping of human natural killer cells.

This protocol details the step-by-step procedure for in-depth immune phenotyping of peripheral blood natural killer (NK) cells from clinical samples by mass cytometry. The protocol consists of three main steps: PBMC incubation with a mix of metal-conjugated antibodies for extracellular phenotyping followed by fixation, permeabilization and incubation with a mix of metal-conjugated antibodies for staining of intracellular proteins, and sample acquisition on a mass cytometer. High-dimensional analysis enables the visualization of NK cell subsets and their phenotypical characteristics. For complete details on the use and execution of this protocol, please refer to Chretien et al. (2021).

TET2 regulates immune tolerance in chronically activated mast cells.

Mutation of the TET2 DNA-hydroxymethylase has been associated with a number of immune pathologies. The disparity in phenotype and clinical presentation among these pathologies leads to questions regarding the role of TET2 mutation in promoting disease evolution in different immune cell types. Here we show that, in primary mast cells, Tet2 expression is induced in response to chronic and acute activation signals. In TET2-deficient mast cells, chronic activation via the oncogenic KITD816V allele associated with mastocytosis, selects for a specific epigenetic signature characterized by hypermethylated DNA regions (HMR) at immune response genes. H3K27ac and transcription factor binding is consistent with priming or more open chromatin at both HMR and non-HMR in proximity to immune genes in these cells, and this signature coincides with increased pathological inflammation signals. HMR are also associated with a subset of immune genes that are direct targets of TET2 and repressed in TET2-deficient cells. Repression of these genes results in immune tolerance to acute stimulation that can be rescued with vitamin C treatment or reiterated with a Tet inhibitor. Overall, our data support a model where TET2 plays a direct role in preventing immune tolerance in chronically activated mast cells, supporting TET2 as a viable target to reprogram the innate immune response for innovative therapies.

PD-L1 and PD-L2 differ in their molecular mechanisms of interaction with PD-1.

The programmed death-1 (PD-1) molecule is involved in peripheral tolerance and in the immune escape mechanisms during chronic viral infections and cancer. PD-1 interacts with two ligands, PD-L1 and PD-L2. We have investigated the molecular mechanisms of PD-1 interactions with its ligands by surface plasmon resonance and cell surface binding as well as the ability of the two ligands to compete for PD-1 binding. PD-L1 and PD-L2 bound PD-1 with comparable affinities, but striking differences were observed at the level of the association and dissociation characteristics. PD-L1, but not PD-L2, had a delayed interaction reminiscent of a phenomenon of conformational transition. These mechanisms were confirmed by using PD-L1 mAbs that delayed the dissociation of PD-L1 from PD-1. This mechanism was not restricted to PD-1 binding since PD-L1 behaved in a similar manner with its second ligand, CD80. Finally, we could demonstrate that PD-L1 and PD-L2 competed for PD-1 binding and conversely, an antagonist PD-1 mAb blocked both PD-L1 and PD-L2 binding to PD-1 and strongly enhanced T-cell proliferation. These data further emphasize the differential molecular mechanisms of interaction of PD-L1 and PD-L2 with PD-1, and suggest possible new approach for the therapy of chronic infection, cancer and transplantation.

Cutting edge: Dok-1 and Dok-2 adaptor molecules are regulated by phosphatidylinositol 5-phosphate production in T cells.

Downstream of tyrosine kinase (Dok) proteins Dok-1 and Dok-2 are involved in T cell homeostasis maintenance. Dok protein tyrosine phosphorylation plays a key role in establishing negative feedback loops of T cell signaling. These structurally related adapter molecules contain a pleckstrin homology (PH) domain generally acting as a lipid/protein-interacting module. We show that the presence of this PH domain is necessary for the tyrosine phosphorylation of Dok proteins and their negative functions in T cells. We find that Dok-1/Dok-2 PH domains bind in vitro to the rare phosphoinositide species, phosphatidylinositol 5-phosphate (PtdIns5P). Dok tyrosine phosphorylation correlates with PtdIns5P production in T cells upon TCR triggering. Furthermore, we demonstrate that PtdIns5P increase regulates Dok tyrosine phosphorylation in vivo. Together, our data identify a novel lipid mediator in T cell signaling and suggest that PH-PtdIns5P interactions regulate T cell responses.