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)).

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.

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).

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.

CD47-SIRPα Controls ADCC Killing of Primary T Cells by PMN Through a Combination of Trogocytosis and NADPH Oxidase Activation.

Immunotherapies targeting the "don't eat me" myeloid checkpoint constituted by CD47 SIRPα interaction have promising clinical potential but are limited by toxicities associated with the destruction of non-tumor cells. These dose-limiting toxicities demonstrate the need to highlight the mechanisms of anti-CD47-SIRPα therapy effects on non-tumor CD47-bearing cells. Given the increased incidence of lymphopenia in patients receiving anti-CD47 antibodies and the strong ADCC (antibody-dependent cellular cytotoxicity) effector function of polymorphonuclear cells (PMNs), we investigated the behavior of primary PMNs cocultured with primary T cells in the presence of anti-CD47 mAbs. PMNs killed T cells in a CD47-mAb-dependent manner and at a remarkably potent PMN to T cell ratio of 1:1. The observed cytotoxicity was produced by a novel combination of both trogocytosis and a strong respiratory burst induced by classical ADCC and CD47-SIRPα checkpoint blockade. The complex effect of the CD47 blocking mAb could be recapitulated by combining its individual mechanistic elements: ADCC, SIRPα blockade, and ROS induction. Although previous studies had concluded that disruption of SIRPα signaling in PMNs was limited to trogocytosis-specific cytotoxicity, our results suggest that SIRPα also tightly controls activation of NADPH oxidase, a function demonstrated during differentiation of immature PMNs but not so far in mature PMNs. Together, our results highlight the need to integrate PMNs in the development of molecules targeting the CD47-SIRPα immune checkpoint and to design agents able to enhance myeloid cell function while limiting adverse effects on healthy cells able to participate in the anti-tumor immune response.

Targeting CISH enhances natural cytotoxicity receptor signaling and reduces NK cell exhaustion to improve solid tumor immunity.

BACKGROUND: The success and limitations of current immunotherapies have pushed research toward the development of alternative approaches and the possibility to manipulate other cytotoxic immune cells such as natural killer (NK) cells. Here, we targeted an intracellular inhibiting protein 'cytokine inducible SH2-containing protein' (CISH) in NK cells to evaluate the impact on their functions and antitumor properties. METHODS: To further understand CISH functions in NK cells, we developed a conditional Cish-deficient mouse model in NK cells (Cishfl/flNcr1Ki/+ ). NK cells cytokine expression, signaling and cytotoxicity has been evaluated in vitro. Using intravenous injection of B16F10 melanoma cell line and EO711 triple negative breast cancer cell line, metastasis evaluation was performed. Then, orthotopic implantation of breast tumors was performed and tumor growth was followed using bioluminescence. Infiltration and phenotype of NK cells in the tumor was evaluated. Finally, we targeted CISH in human NK-92 or primary NK cells, using a technology combining the CRISPR(i)-dCas9 tool with a new lentiviral pseudotype. We then tested human NK cells functions. RESULTS: In Cishfl/flNcr1Ki/+ mice, we detected no developmental or homeostatic difference in NK cells. Global gene expression of Cishfl/flNcr1Ki/+ NK cells compared with Cish+/+Ncr1Ki/+ NK cells revealed upregulation of pathways and genes associated with NK cell cycling and activation. We show that CISH does not only regulate interleukin-15 (IL-15) signaling pathways but also natural cytotoxicity receptors (NCR) pathways, triggering CISH protein expression. Primed Cishfl/flNcr1Ki/+ NK cells display increased activation upon NCR stimulation. Cishfl/flNcr1Ki/+ NK cells display lower activation thresholds and Cishfl/flNcr1Ki/+ mice are more resistant to tumor metastasis and to primary breast cancer growth. CISH deletion favors NK cell accumulation to the primary tumor, optimizes NK cell killing properties and decreases TIGIT immune checkpoint receptor expression, limiting NK cell exhaustion. Finally, using CRISPRi, we then targeted CISH in human NK-92 or primary NK cells. In human NK cells, CISH deletion also favors NCR signaling and antitumor functions. CONCLUSION: This study represents a crucial step in the mechanistic understanding and safety of Cish targeting to unleash NK cell antitumor function in solid tumors. Our results validate CISH as an emerging therapeutic target to enhance NK cell immunotherapy.

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.

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.

Quantification of Immune Variables from Liquid Biopsy in Breast Cancer Patients Links Vδ2+ γδ T Cell Alterations with Lymph Node Invasion.

The rationale for therapeutic targeting of Vδ2+ γδ T cells in breast cancer is strongly supported by in vitro and murine preclinical investigations, characterizing them as potent breast tumor cell killers and source of Th1-related cytokines, backing cytotoxic αß T cells. Nonetheless, insights regarding Vδ2+ γδ T cell phenotypic alterations in human breast cancers are still lacking. This paucity of information is partly due to the challenging scarcity of these cells in surgical specimens. αß T cell phenotypic alterations occurring in the tumor bed are detectable in the periphery and correlate with adverse clinical outcomes. Thus, we sought to determine through an exploratory study whether Vδ2+ γδ T cells phenotypic changes can be detected within breast cancer patients' peripheral blood, along with association with tumor progression. By using mass cytometry, we quantified 130 immune variables from untreated breast cancer patients' peripheral blood. Supervised analyses and dimensionality reduction algorithms evidenced circulating Vδ2+ γδ T cell phenotypic alterations already established at diagnosis. Foremost, terminally differentiated Vδ2+ γδ T cells displaying phenotypes of exhausted senescent T cells associated with lymph node involvement. Thereby, our results support Vδ2+ γδ T cells implication in breast cancer pathogenesis and progression, besides shedding light on liquid biopsies to monitor surrogate markers of tumor-infiltrating Vδ2+ γδ T cell antitumor activity.

Chronic IL-15 Stimulation and Impaired mTOR Signaling and Metabolism in Natural Killer Cells During Acute Myeloid Leukemia.

Natural Killer (NK) cells are potent anti-leukemic immune effectors. However, they display multiple defects in acute myeloid leukemia (AML) patients leading to reduced anti-tumor potential. Our limited understanding of the mechanisms underlying these defects hampers the development of strategies to restore NK cell potential. Here, we have used a mouse model of AML to gain insight into these mechanisms. We found that leukemia progression resulted in NK cell maturation defects and functional alterations. Next, we assessed NK cell cytokine signaling governing their behavior. We showed that NK cells from leukemic mice exhibit constitutive IL-15/mTOR signaling and type I IFN signaling. However, these cells failed to respond to IL-15 stimulation in vitro as illustrated by reduced activation of the mTOR pathway. Moreover, our data suggest that mTOR-mediated metabolic responses were reduced in NK cells from AML-bearing mice. Noteworthy, the reduction of mTOR-mediated activation of NK cells during AML development partially rescued NK cell metabolic and functional defects. Altogether, our data strongly suggest that NK cells from leukemic mice are metabolically and functionally exhausted as a result of a chronic cytokine activation, at least partially IL-15/mTOR signaling. NK cells from AML patients also displayed reduced IL-2/15Rß expression and showed cues of reduced metabolic response to IL-15 stimulation in vitro, suggesting that a similar mechanism might occur in AML patients. Our study pinpoints the dysregulation of cytokine stimulation pathways as a new mechanism leading to NK cell defects in AML.

NK Cell Priming From Endogenous Homeostatic Signals Is Modulated by CIS.

Natural killer (NK) cell activation is controlled by a balance of activating and inhibitory signals and cytokines such as IL-15. We previously identified cytokine-inducible SH2-containing protein (CIS) as a negative regulator of IL-15 signaling in NK cells under inflammatory conditions. While the functional effect of Cish-deficiency in NK cells was obvious by their increased anti-tumor immunity and hyper-proliferative response to IL-15, it remained unclear how CIS regulates NK cell biology in steady-state. Here, we investigated the role of CIS in the homeostatic maintenance of NK cells and found CIS-ablation promoted terminal differentiation of NK cells and increased turnover, suggesting that under steady-state conditions, CIS plays a role in maintaining IL-15 driven regulation of NK cells in vivo. However, hyper-responsiveness to IL-15 did not manifest in NK cell accumulation, even when the essential NK cell apoptosis mediator, Bcl2l11 (BIM) was deleted in addition to Cish. Instead, loss of CIS conferred a lower activation threshold, evidenced by augmented functionality on a per cell basis both in vitro and in vivo without prior priming. We conclude that Cish regulates IL-15 signaling in NK cells in vivo, and through the rewiring of several activation pathways leads to a reduction in activation threshold, decreasing the requirement for priming and improving NK cell anti-tumor function. Furthermore, this study highlights the tight regulation of NK cell homeostasis by several pathways which prevent NK cell accumulation when IL-15 signaling and intrinsic apoptosis are dysregulated.

Vitamin D Controls Tumor Growth and CD8+ T Cell Infiltration in Breast Cancer.

Women with low levels of vitamin D have a higher risk of developing breast cancer. Numerous studies associated the presence of a CD8+ T cell infiltration with a good prognosis. As vitamin D may play a key role in the modulation of the immune system, the objective of this work was to evaluate the impact of vitamin D on the breast cancer progression and mammary tumor microenvironment. We show that vitamin D decreases breast cancer tumor growth. Immunomonitoring of the different immune subsets in dissociated tumors revealed an increase in tumor infiltrating CD8+ T cells in the vitamin D-treated group. Interestingly, these CD8+ T cells exhibited a more active T cell (TEM/CM) phenotype. However, in high-fat diet conditions, we observed an opposite effect of vitamin D on breast cancer tumor growth, associated with a reduction of CD8+ T cell infiltration. Our data show that vitamin D is able to modulate breast cancer tumor growth and inflammation in the tumor microenvironment in vivo. Unexpectedly, this effect is reversed in high-fat diet conditions, revealing the importance of diet on tumor growth. We believe that supplementation with vitamin D can in certain conditions represent a new adjuvant in the treatment of breast cancers.

TCR and CD28 Concomitant Stimulation Elicits a Distinctive Calcium Response in Naive T Cells.

T cell activation is initiated upon ligand engagement of the T cell receptor (TCR) and costimulatory receptors. The CD28 molecule acts as a major costimulatory receptor in promoting full activation of naive T cells. However, despite extensive studies, why naive T cell activation requires concurrent stimulation of both the TCR and costimulatory receptors remains poorly understood. Here, we explore this issue by analyzing calcium response as a key early signaling event to elicit T cell activation. Experiments using mouse naive CD4+ T cells showed that engagement of the TCR or CD28 with the respective cognate ligand was able to trigger a rise in fluctuating calcium mobilization levels, as shown by the frequency and average response magnitude of the reacting cells compared with basal levels occurred in unstimulated cells. The engagement of both TCR and CD28 enabled a further increase of these two metrics. However, such increases did not sufficiently explain the importance of the CD28 pathways to the functionally relevant calcium responses in T cell activation. Through the autocorrelation analysis of calcium time series data, we found that combined but not separate TCR and CD28 stimulation significantly prolonged the average decay time (τ) of the calcium signal amplitudes determined with the autocorrelation function, compared with its value in unstimulated cells. This increasement of decay time (τ) uniquely characterizes the fluctuating calcium response triggered by concurrent stimulation of TCR and CD28, as it could not be achieved with either stronger TCR stimuli or by co-engaging both TCR and LFA-1, and likely represents an important feature of competent early signaling to provoke efficient T cell activation. Our work has thus provided new insights into the interplay between the TCR and CD28 early signaling pathways critical to trigger naive T cell activation.

T Cells on Engineered Substrates: The Impact of TCR Clustering Is Enhanced by LFA-1 Engagement.

We created APC-mimetic synthetic substrates to study the impact of ligand clustering on T cell activation and spreading. The substrates exhibit antibodies directed against the TCR-complex in the form of a patterned array of sub micrometric dots surrounded by a fluid supported lipid bilayer (SLB) which may itself be functionalized with another bio-molecule. We show that for T cell adhesion mediated by T cell receptor (TCR) alone, in the patterned, but not in the corresponding homogeneous controls, the TCR, ZAP-70 and actin are present in the form of clusters or patches that co-localize with the ligand-dots. However, global cell scale parameters like cell area and actin distribution are only weakly impacted by ligand clustering. In presence of ICAM-1 - the ligand of the T cell integrin LFA-1 - on the SLB, the TCR is still clustered due to the patterning of its ligands, but now global parameters are also impacted. The actin organization changes to a peripheral ring, resembling the classical actin distribution seen on homogeneous substrates, the patterned membrane topography disappears and the membrane is flat, whereas the cell area increases significantly. These observations taken together point to a possible pivotal role for LFA-1 in amplifying the effect of TCR-clustering. No such effect is evident for co-engagement of CD28, affected via its ligand B7.2. Unlike on ICAM-1, on B7.2 cell spreading and actin organization are similar for homogeneous and patterned substrates. However, TCR and ZAP-70 clusters are still formed in the patterned case. These results indicate complementary role for LFA-1 and CD28 in the regulation and putative coupling of TCR micro-clusters to actin. The engineered substrates presented here clearly have the potential to act as platform for fundamental research in immune cell biology, as well as translational analyses in immunotherapy, for example to screen molecules for their role in T cell adhesion/activation.