Search

International audience; No abstract available

Purpose: Immune checkpoint inhibitors (ICI) have revolutionized the treatment of non–small cell lung cancer (NSCLC), but predictive biomarkers of their efficacy are imperfect. The primary objective is to evaluate circulating immune predictors of pembrolizumab efficacy in patients with advanced NSCLC. Experimental Design: We used high-dimensional mass cytometry (CyTOF) in baseline blood samples of patients with advanced NSCLC treated with pembrolizumab. CyTOF data were analyzed by machine-learning algorithms (Citrus, tSNE) and confirmed by manual gating followed by principal component analysis (between-group analysis). Results: We analyzed 27 patients from the seminal KEYNOTE-001 study (median follow-up of 60.6 months). We demonstrate that blood baseline frequencies of classical monocytes, natural killer (NK) cells, and ICOS+ CD4+ T cells are significantly associated with improved objective response rates, progression-free survival, and overall survival (OS). In addition, we report that a baseline immune peripheral score combining these three populations strongly predicts pembrolizumab efficacy (OS: HR = 0.25; 95% confidence interval = 0.12–0.51; P < 0.0001). Conclusions: As this immune monitoring is easy in routine practice, we anticipate our findings may improve prediction of ICI benefit in patients with advanced NSCLC.

The success of immunotherapy has highlighted the critical role of the immune microenvironment in acute lymphoblastic leukemia (ALL); however, the immune landscape in ALL remains incompletely understood and most studies have focused on conventional T cells or NK cells. This study investigated the prognostic impact of circulating γδ T-cell alterations using high-dimensional analysis in a cohort of newly diagnosed adult ALL patients (10 B-ALL; 9 Philadelphia+ ALL; 9 T-ALL). Our analysis revealed common alterations in CD8+ T cells and γδ T cells of relapsed patients, including accumulation of early stage differentiation and increased expression of BTLA and CD73. We demonstrated that the circulating γδ T-cell signature was the most discriminating between relapsed and disease-free groups. In addition, Vδ2 T-cell alterations strongly discriminated patients by relapse status. Taken together, these data highlight the role of ɣδ T cells in adult ALL patients, among whom Vδ2 T cells may be a pivotal contributor to T-cell immunity in ALL. Our findings provide a strong rationale for further monitoring and potentiating Vδ2 T cells in ALL, including in the autologous setting.

Purpose:Immune checkpoint inhibitors (ICI) have revolutionized the treatment of non–small cell lung cancer (NSCLC), but predictive biomarkers of their efficacy are imperfect. The primary objective is to evaluate circulating immune predictors of pembrolizumab efficacy in patients with advanced NSCLC. Experimental Design:We used high-dimensional mass cytometry (CyTOF) in baseline blood samples of patients with advanced NSCLC treated with pembrolizumab. CyTOF data were analyzed by machine-learning algorithms (Citrus, tSNE) and confirmed by manual gating followed by principal component analysis (between-group analysis).Results:We analyzed 27 patients from the seminal KEYNOTE-001 study (median follow-up of 60.6 months). We demonstrate that blood baseline frequencies of classical monocytes, natural killer (NK) cells, and ICOS+ CD4+ T cells are significantly associated with improved objective response rates, progression-free survival, and overall survival (OS). In addition, we report that a baseline immune peripheral score combining these three populations strongly predicts pembrolizumab efficacy (OS: HR = 0.25; 95% confidence interval = 0.12–0.51; P < 0.0001).Conclusions:As this immune monitoring is easy in routine practice, we anticipate our findings may improve prediction of ICI benefit in patients with advanced NSCLC.

Supplementary Figure 1: Graphical Abstract Supplementary Figure 2: Overall Survival in NSCLC patients treated with pembrolizumab in KEYNOTE-001 according to EGFR mutation Supplementary Figure 3: Recursive partitioning identified classical monocytes and NK as the main immune populations whose frequency splits patients into distinct patterns of overall survival Supplementary Figure 4: Classical monocytes, NK cells and ICOS+ CD4+ T cells are associated with improved pembrolizumab efficacy in advanced NSCLC patients. Supplementary Figure 5: Gating strategy of the 3 main immune populations associated with pembrolizumab efficacy. Supplementary Figure 6: Comparison side by side of Maxstat cut-offs with cut-off from Younden index for overall survival in the 3 populations of interest. Supplementary Figure 7: Survival outcomes in NSCLC patients treated with pembrolizumab in KEYNOTE-001 according to blood baseline frequency of classical monocytes. Supplementary Figure 8: Baseline immune peripheral score is also associated with PFS in melanoma patients treated with PD-1 inhibitors.

The induction of proinflammatory T cells by dendritic cell (DC) subtypes is critical for antitumor responses and effective immune checkpoint blockade (ICB) therapy. Here, we show that human CD1c + CD5 + DCs are reduced in melanoma-affected lymph nodes, with CD5 expression on DCs correlating with patient survival. Activating CD5 on DCs enhanced T cell priming and improved survival after ICB therapy. CD5 + DC numbers increased during ICB therapy, and low interleukin-6 (IL-6) concentrations promoted their de novo differentiation. Mechanistically, CD5 expression by DCs was required to generate optimally protective CD5 hi T helper and CD8 + T cells; further, deletion of CD5 from T cells dampened tumor elimination in response to ICB therapy in vivo. Thus, CD5 + DCs are an essential component of optimal ICB therapy.

Over the past few decades, with the rise of immunotherapies, tumor infiltrating immune cells were increasingly investigated. Indeed, they may represent biomarkers for patient outcome prediction, they may bear immune checkpoint markers that can be targeted by therapeutic antibodies and mechanistic studies may reveal how to tweak their activation profile so that we can re-direct them towards tumor cells. Macrophages possess a central place in tissue homeostasis for tissue remodeling and cleaning, transformed cell elimination, phagocytosis and regulation of inflammation via cytokine production. All these functions allow the discovery of approaches to target Tumor Associated Macrophages (TAMs) using immunotherapies. Indeed, TAMs express known immune checkpoint markers such as PD-L1, CD40, Sirp-α and markers such as CD163, CD204, TREM2, TREM1 associated with prognosis. In the context of therapies TAM may participate to antibody dependent cell phagocytosis (ADCP) thanks to FCγ-Receptors. Here, we will review the recent literature on TAMs in the specific context of HPV

TNFRF-14/HVEM is the ligand for 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. We developed C57BL/6 mouse models co-expressing human huBTLA and huHVEM as well as antagonistic monoclonal antibodies (mAbs) that completely prevent the interactions of HVEM with its ligands. Here, we show that the anti-HVEM18-10 mAb increases primary human αβ-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 anti-PD-L1 mAb to activate T cells in the presence of PDL-1 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 effect in vivo and especially disentangle its CIS and TRANS effects, we developed a knock-in (KI) mouse model expressing human BTLA (huBTLA+/+) and a KI mouse model expressing both human BTLA and human HVEM (huBTLA+/+/huHVEM+/+ (DKI)). In vivo pre-clinical experiments performed in both mouse models showed that HVEM18-10 treatment was efficient to decrease human HVEM+ tumor growth. In the DKI model, anti-HVEM 18-10 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 upon re-challenge in both settings, therefore showing a marked T cell-memory phenotype effect. 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 (anti-PD1/anti-PDL-1/anti-CTLA-4). Citation Format: Laurent GORVEL, Clemence Demerle, Marielle Mello, Sonia Pastor, Clara Degos, Ana Zarubica, Fabien Angelis, Frederic Fiore, Jacques Nunes, Bernard Malissen, Laurent Greillier, Geoffrey Guittard, Herve Luche, Fabrice Barlesi, Daniel Olive. 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. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5184.

Background: PD-1 blockade (αPD-1) has shown limited efficacy in breast & gynecologic cancers. In other types of solid tumor, regulatory T cells (Tregs) harbouring markers of highly suppressive effector Tregs (eTregs) correlates with poor responses to PD-1 blockade, prompting combination therapy based on eTregs depletion. Yet, subsets of Tregs remain to be determined in breast & gynecologic cancers, in order to: (i) evaluate the role of eTregs in resistances to PD-1 blockade, (ii) identify the most selective target for eTregs depletion tailored to tumors context and combination strategy. Methods: We collected public single-cell RNA/TCR-sequencing data from primary tumors and metastases of Triple-Negative Breast Cancer patients (TNBC, N = 28) biopsied before and after αPD-1 (pembrolizumab). Microarray data from primary TNBC (N = 124) biopsied before and after αPD-1 were quired for association with clinical responses. Deep phenotyping of Tregs from normal breast tissues (N = 4) and primary breast tumors (N = 8) or gynecologic tumors (N = 17) was performed by mass cytometry. ALD2510 (αCD25NIB, Alderaan Biotechnology) is a novel non-IL-2 blocking Fc-optimized anti-CD25 mAb (1). αPD-1 and αCD25NIB combination was evaluated using: (i) a humanized αCD25NIB in CD34+ humanized NSG mice grafted with human TNBC cell lines, (ii) a murine surrogate of αCD25NIB in a syngeneic TNBC mouse model. Results: CD25high Tregs were accumulating in breast & gynecologic tumor tissues. In TNBC patients treated with αPD-1, fractions of CD25high Tregs were further increased. CD25high Tregs highly expressed eTregs-related molecules in primary tumors, invaded tumor-draining lymph nodes and distant metastases. Amongst the potential therapeutic targets for Treg depletion, only CD25, 4-1BB and CCR8 were largely restricted to intratumoral CD25high eTregs. Yet, CD25 was the best candidate for CD25high eTregs depletion with limited on-target off-Treg effects, as: (i) CCR8 marked a small fractions of CD25high eTregs in primary tumors and was not detected in metastasis, (ii) 4-1BB marked antigen-experienced αβCD8 T cell revigorated by PD-1 blockade, (iii) only CD25high eTregs expressed 4-1BB and CCR8, yet TCR clonotype analysis revealed that CD25high eTregs originate in part from activated CD25+ Tregs, suggesting that anti-CCR8/-4-1BB mAbs may be ineffective due to CD25high eTregs replenishment in tumors. In murine models resistant to αPD-1 alone, αCD25NIB effectively depleted intratumoral CD25high eTregs, with limited effects on peripheral Tregs, and synergized with αPD-1 by restoring a positive effector αβCD8 T cells/Tregs ratio, leading to tumor clearance without adverse effects. Conclusions: This study supports clinical evaluation of CD25high effector Tregs depletion by ALD2510 in patients with breast or gynecologic cancers resistant to PD-1 blockade. Citation Format: Stéphane FATTORI, Aude Le Roy, Jemila Houacine, Lucie Robert, Riad Abes, Laurent Gorvel, Samuel Granjeaud, Marie-Sarah Rouvière, Amira Ben Amara, Nicolas Boucherit, Carole Tarpin, Jihane Pakradouni, Emmanuelle Charafe-Jauffret, Julien Barrou, Gilles Houvenaeghel, Eric Lambaudie, François Bertucci, Philippe Rochigneux, Anthony Gonçalves, Arnaud Foussat, Anne-Sophie Chrétien, Daniel Olive. Selective depletion of regulatory T cells by ALD2510, a novel IL-2-sparing anti-CD25 antibody, synergizes with PD-1 blockade in breast and gynecologic cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3239.

Within the tumor microenvironment (TME), TREGS have been associated with worse prognosis and/or resistance to checkpoint inhibitor (CPI) therapy in various cancer diseases, making these cells a promising target for the development of new immunotherapies. Here, we present recent preclinical development data about ALD2510, a next-gen low-fucose IL-2/TCONV-sparing anti-CD25 antibody designed for the selective depletion of CD25high TREGS and the boost of host anti-tumor immunity. Ultra-humanization of parental anti-CD25 antibody was achieved through phage display and, in parallel, all putative liability sequences were replaced for maximizing developability while minimizing off-target and immunogenicity risks. ALD2510 sequences were re-formatted and used for further CHO cell line development, using the GlymaxX™ technology which boosts Fc effector functions. Early tox ALD2510 material was produced following 10L fed-batch USP/DSP and analytical characterization, including a 6-month early stability study. Preliminary safety and pharmacokinetic/pharmacodynamic (PKPD) profile of ALD2510 were then determined through a non-GLP MTD/DRF study in cynomolgus monkey. ALD2510 demonstrated excellent potency (TREG depletion, ADCC, ADCP), manufacturability and stability together with very low immunogenicity potential. CHO productivity reached ~4g/L in 10L fed-batch bioreactor and characterization revealed excellent purity and activity in line with its low fucose content, together with very low levels of process- or cell-related impurities. This material showed a good behavior during the 6-month stability study and was therefore administered in cynomolgus monkeys during a non-GLP exploratory MTD/DRF study. Increasing doses of ALD2510 (from 0.3 to 100mg/kg) were given to four animals during MTD and same animals received three additional doses at 100mg/kg during DRF. Very good tolerability and safety were reported in animals. ALD2510 half-life and exposure were found consistent with that of humanized IgG1 in cynomolgus. Strong TREG depletion was monitored in the blood in all animals with, importantly, no drop was observed in CD4+ or CD8+ T-cells, confirming ALD2510 capacity to selectively target TREGS while sparing TCONV. Overall, ALD2510 demonstrated excellent potency, manufacturability and stability during the 1st phase of preclinical development. Very good tolerability and safety profile together with satisfying PKPD data were reported during exploratory MTD/DRF study in cynomolgus monkeys. Noteworthy, strong TREG depletion was confirmed while CD4+ or CD8+ T-cells were not impacted, confirming ALD2510 selectivity for TREGS while sparing TCONV. This makes ALD2510 a promising candidate for the treatment of solid tumors, in particular those where the presence of TREGS in the TME is associated with worse prognosis and/or resistance to CPI. Citation Format: Jemila Houacine, Riad Abes, Anne Marie-Cardine, Aude Le Roy, Bettina Serbin, Lucie Robert, Jérôme Giustiniani, Anne-Sophie Chrétien, Stéphane Fattori, Laurent Gorvel, Armand Bensussan, Daniel Olive, Arnaud Foussat. Preclinical development of ALD2510, a Next-Gen Fc-enhanced, TREG-selective and IL-2-sparing anti-CD25 antibody for the treatment of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 710.

Vγ9Vδ2 T cells play a key role in the innate immune response to viral infections, including SARS-CoV-1 and 2, and are activated through butyrophilin (BTN)-3A. Here, the objectives were to: 1) characterize the effects of SARS-CoV-2 infection on the number, phenotype, and activation of Vγ9Vδ2 T cells in infected patients, and 2) assess the effects of in vitro SARS-CoV-2 infection on the expression of BTN3A and its impact on the activation and response of Vγ9Vδ2 T cells to an anti-BTN3A antibody. Blood Vγ9Vδ2 T cells decreased in clinically mild SARS-CoV-2 infections compared to healthy volunteers (HV). This decrease was maintained up to 28 days and in the recovery period. Terminally differentiated Vγ9Vδ2 T cells tend to be enriched on the day of diagnosis, 28 days after and during the recovery period compared to HV. Furthermore, these cells showed cytotoxic and inflammatory activities as shown by TNFα, IFNγ and CD107a/b increase following anti-BTN3A activation. Moreover, BTN3A upregulation and Vγ9Vδ2 T cell infiltration were observed in a lung biopsy from a fatal SARS-CoV-2 infection, as compared to HV. In vitro, SARS-CoV-2 infection significantly increased BTN3A expression in macrophages and lung cell lines. The activation via BTN3A enhanced the anti-SARS-CoV-2 Vγ9Vδ2 T cells cytotoxicity and IFN-γ and TNFα in SARS-CoV-2 infected patient. Increasing concentrations of anti-BTN3A were accompanied by an inhibition of viral replication. Altogether, these data suggest that Vγ9Vδ2 T cells are important in the immune response against SARS-CoV-2 infection and that activation by an anti-BTN3A antibody may enhance their response.KEY POINTSSARS-CoV-2 mediates upregulation of the key receptor of Vγ9Vδ2 T cells BTN3A on lung tissues and cell lines as well as monocytesDuring SARS-CoV-2 infection, Vγ9Vδ2 are differentiated and efficiently degranulate and secrete cytokines upon activation with BTN3A mAb

Regulatory T cells (Treg) are an immunosuppressive subtype of CD4+ T cells essential for maintaining self-tolerance in physiological settings. Tregs also abundantly infiltrate inflamed tumor tissues, impeding the host's antitumor immune response and contributing to tumor growth and metastasis. In breast cancers, subsets of Tregs express highly immunosuppressive effector phenotypes that favor tumorigenesis, progression, and resistance to immune-checkpoint inhibitor therapies. Tregs share phenotypic features with cytotoxic lymphocytes, rendering them difficult to inhibit without compromising productive antitumor immunity. In addition, systemic targeting of Tregs causes serious autoimmune adverse events in patients with cancer. Hence, the identification of candidate targets or methodologies allowing the specific elimination of tumor antigen-specific Tregs, including tumor-infiltrating Tregs, is a prerequisite for developing efficient and safe combinatorial immunotherapeutic strategies in breast cancers. To date, numerous preclinical studies have demonstrated that specific targeting of breast tumor–infiltrating Tregs restores a competent antitumor immune response and improves responses to immune-checkpoint inhibitors such as PD-1/PD-L1 blockade. Herein, we discuss major candidate molecules for Treg-targeted therapeutic strategies in breast cancers, detailing the pros and cons of various approaches, including mAb-mediated depletion, homeostasis destabilization, and functional blockade.

Background: Advanced cutaneous T-cell lymphomas (CTCLs) remain an unmet medical need. Brentuximab vedotin (BV), an anti-CD30 antibody-drug conjugate (ADC) linked to monomethyl auristatin E (MMAE), do not deliver significant long-term improvements in patient outcomes. More recently, mogamulizumab and anti-KIR3DL2 provided encouraging results but new targeted therapies are needed. Inducible Co-Stimulator (ICOS), a T-cell costimulatory receptor involved in the development of CTCLs, arouses interest. Methods: We used immunohistochemistry to study ICOS expression in skin biopsies of 23 patients with early-stage mycosis fungoides (MF), 12 with transformed MF (TMF) and 17 with Sézary Syndrome (SS), at diagnosis or in relapse. Skin samples from 12 patients with B-cell lymphomas, 14 with CD30 + lymphoproliferative disease (LPD), 12 with primary cutaneous CD4+ small/medium T-cell lympho-proliferative disorder and 13 with angioimmunoblastic T-cell lymphoma (AITL) were used as controls. ICOS expression by circulating Sézary cells and regulatory T cells (Tregs) in patients with SS was evaluated using flow cytometry and compared to healthy donors (HD) lymphocytes. In 5 patients with SS, we also analyzed concomitant biopsies from involved nodes. Then, we investigated the efficacy of anti-ICOS ADCs generated by coupling murine anti-ICOS 314.8 monoclonal antibodies with MMAE and pyrrolobenzodiazepine (PBD), in comparison to BV. We used ICOS + CTCL cell lines (MyLa, MJ and HUT78), murine xenograft models with MyLa and ICOS + Patient Derived Xenografts (PDXs) from patients with SS and AITL. In order to identify the best anti-ICOS clone that we should develop for a clinical trial, we evaluated the affinity of the antibody on the receptor, the internalization capacity of the antibody using pHAb Reactive Dyes kit (Promega®), and the ability of the antibody to act as an ADC using a secondary conjugate (Mab-ZAP kit, Advanced Targeting Systems®). Results: ICOS was highly expressed by the cutaneous atypical lymphocytic infiltrates in respectively 61%, 75% and 88% of patients with early-stage MF, TMF and SS, such as in all the involved nodes. Double staining experiments which were performed in both skin and lymph node revealed that ICOS expression appears mainly restricted to neoplastic CD4 + T-cells, with rare ICOS +CD8 + T-cells in the tumor micro-environment. ICOS expression by circulating Sézary cells was strong: 69 ± 7.3% versus 38.8 ± 7.1% of non-tumoral CD4 + cells (p In CTCL cell lines, we observed a significant dose-dependent decrease in cell viability in the presence of anti-ICOS-MMAE (IC50 = 8.2ng/mL) and anti-ICOS-PBD (IC50 = 1.2ng/mL) ADCs. In a mouse xenograft model (MyLa), anti-ICOS-MMAE ADCs provided a longer overall survival (OS) than BV (HR=15.2; CI95%: 3.2-71.1; p Among 8 different anti-ICOS clones, clone 314.8 had the best affinity on MyLa and MJ cell lines. Clones 53.3, 293.1, 92.17, 88.2 and 279.1 also had good affinity to receptor, whereas clones 145.1 and 121.4 had poor affinity. Using the internalization pHAb reactive dyes kit, we found that clones 314.8, 53.3, 92.17, 88.2 internalized significantly better and faster than the other ones. In order to verify if there is a correlation between internalization capacity and ADC effect, clones 53.3, 92.17 and 145.1 were coupled to MMAE. While anti-ICOS-53.3 and anti-ICOS-92.17 ADCs had similar efficacy to anti-ICOS-314.8 ADCs on MyLa, anti-ICOS 145.1 ADCs resulted in significantly lower cell death. Finally, all clones showed good ability to act as ADCs with Mab-ZAP, excluding clones 279.1, 145.1 and 121.4. Discussion: ICOS is a promising therapeutic target because it is expressed both by tumor T-cells and regulatory T-cells. We report for the first time the strong and frequent expression of ICOS in CTCLs, as well as the preclinical efficacy of anti-ICOS ADCs on CTCL cell line and PDXs. These results could be extended to the spectrum of follicular variant peripheral T-cell lymphomas. Conclusion: Collectively, our findings provide the preliminary basis for a therapeutic trial Figure 1 Figure 1. Disclosures Lopez: Emergence Therapeutics: Current holder of individual stocks in a privately-held company. Bagot: Takeda: Membership on an entity's Board of Directors or advisory committees. Olive: ImCheck Therapeutics: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Emergence Therapeutics: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Alderaan Biotechnology: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees.

Significance This work provides a report of accumulation of unconventional CD56−CD16+ NK cells in nonvirally induced malignancies. Increased frequency of CD56−CD16+ NK cells is associated with adverse clinical outcome in AML, as well as other maturation defects, and might contribute to a defective control of AML progression. Pseudotime analysis highlights a disruption in the maturation process of conventional NK cells in AML patients, leading to a bifurcation point absent in healthy subjects. This analysis, combined with the reduced frequency of conventional NK cells observed in AML patients, suggests that unconventional CD56−CD16+ NK cells derive from an aberrant maturation of conventional NK cells. Overall, accumulation of CD56−CD16+ NK cells could be an important feature of immune escape from innate immunity., 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.

Introduction Les anti-IL-5, inhibiteurs de la voie Th2, ont recemment ete developpes dans le traitement de l’asthme severe. Nous rapportons le premier cas de syndrome de Sezary (SS) apparu sous benralizumab. Materiel et methodes Un patient de 74 ans etait traite par benralizumab pour un asthme severe. Un mois apres son introduction, il developpait une erythrodermie associee a une keratodermie palmoplantaire. Histologiquement, il s’agissait d’une infiltration cutanee dermique de cellules lymphoides de taille petite a moyenne, atypiques, au noyau irregulier, et avec epidermotropisme discret. L’immunophenotypage sanguin objectivait une hyperlymphocytose TCD4 comprenant 20% de cellules T atypiques CD7+, CD26− et CD158k+, soit 0,6G/L de cellules de Sezary, avec un ratio CD4/CD8 augmente a 6 et un pic monoclonal T identique dans le sang et la peau. Le diagnostic de SS au stade IVB1b etait retenu. Un dosage des cytokines plasmatiques etait realise par Luminex®, en comparant avec le plasma de 2 patients ayant un SS de meme stade au diagnostic, et 2 donneurs sains. Comparativement aux autres patients SS, notre patient presentait des taux eleves d’eotaxine, d’IL-4, d’IL-17 et de CCL3, et un taux faible de CXCL10. Le taux d’IL-5 etait bas sur tous les prelevements. Le patient etait traite par photochimiotherapie extracorporelle et methotrexate, avec une poursuite du benralizumab, permettant un bon controle de son SS et de son asthme. Discussion Le SS est associe a une bascule progressive d’un profil anti-tumoral Th1 a un profil Th2, plus favorable a la proliferation tumorale. L’inhibition de la voie Th2 pourrait donc etre benefique chez les patients ayant un SS, mais notre observation ainsi que des cas rapportes d’aggravation ou de developpement de SS sous dupilumab (anti-IL-4/13) suggerent l’existence de phenomenes paradoxaux. Nous avons observe chez notre patient une elevation franche de l’eotaxine et une elevation plus moderee de l’IL-4, qui sont des acteurs de l’inflammation Th2 dans l’asthme, mais aussi le SS. De meme, des etudes recentes suggerent le role de l’IL-17 dans l’echappement a l’immunite anti-tumorale dans le SS. Par ailleurs, CCL3 est une chimiokine dont le ligand est CCR4, un recepteur des cellules tumorales et des T regulateurs particulierement implique dans le developpement du SS. Enfin, la diminution de CXCL10 pourrait participer a la perte de l’epidermotropisme des cellules de Sezary et a leur circulation dans l’organisme. Toutefois, des etudes dediees sont necessaires pour etablir un lien de causalite entre l’inhibition de l’IL-5 et ces anomalies immunologiques ayant pu participer au developpement du SS chez notre patient. Notre observation rappelle que les consequences immunologiques de l’inhibition therapeutique de voies cytokiniques restent mal connues, et incite a la prudence vis-a-vis de ce type d’effet indesirable a l’heure ou les inhibiteurs de cytokines se multiplient sur le marche.

Simple Summary Vδ2+ γδ T cells have potent antitumor properties both in vitro and in murine preclinical models of breast cancers. However, in the context of human breast cancer, there is a lack of information for potential phenotypic alterations of this crucial immune cell subset. This is partly due to Vδ2+ γδ T cells scarcity in surgical specimens. To break this deadlock, we assessed Vδ2+ γδ T cell phenotypes using untreated breast cancer patients’ peripheral blood, so-called minimally invasive “liquid biopsy”. We show that circulating Vδ2+ γδ T cell phenotypic alterations are already established at diagnosis and related to tumor progression. Notably, terminally differentiated Vδ2+ γδ T cells expressing canonical markers of replicative senescence and exhaustion were significantly associated with tumor-draining lymph node invasion. Our results shed light on the interest of using liquid biopsy to monitor rare events and support Vδ2+ γδ T cell involvement in breast cancer pathogenesis and progression. Abstract 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 αβ 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. αβ 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.

International audience; Endometrial cancer (EC) can easily be cured when diagnosed at an early stage. However, advanced and metastatic EC is a common disease, affecting more than 15,000 patients per year in the United Sates. Only limited treatment options were available until recently, with a taxane–platinum combination as the gold standard in first-line setting and no efficient second-line chemotherapy or hormone therapy. EC can be split into four molecular subtypes, including hypermutated cases with POLE mutations and 25–30% harboring a microsatellite instability (MSI) phenotype with mismatch repair deficiency (dMMR). These tumors display a high load of frameshift mutations, leading to increased expression of neoantigens that can be targeted by the immune system, including (but not limited) to T-cell response. Recent data have demonstrated this impact of programmed death 1 and programmed death ligand 1 (PD-1/PD-L1) inhibitors on chemo-resistant metastatic EC. The uncontrolled KEYNOTE-158 and GARNET trials have shown high response rates with pembrolizumab and dostarlimab in chemoresistant MSI-high tumors. Most responders experiment long responses that last more than one year. Similar, encouraging results were obtained for MMR proficient (MMRp) cases treated with a combination of pembrolizumab and the angiogenesis inhibitor lenvatinib. Approvals have, thus, been obtained or are underway for EC with immune checkpoint inhibitors (ICI) used as monotherapy, and in combination with antiangiogenic agents. Combinations with other targeted therapies are under evaluation and randomized studies are ongoing to explore the impact of ICI-chemotherapy triplets in first-line setting. We summarize in this review the current knowledge of the immune environment of EC, both for MMRd and MMRp tumors. We also detail the main clinical data regarding PD-1/PD-L1 inhibitors and discuss the next steps of development for immunotherapy, including various ICI-based combinations planned to limit resistance to immunotherapy.

International audience; The anti-tumor response of Vγ9Vδ2 T cells requires the sensing of accumulated phosphoantigens (pAgs) bound intracellularly to butyrophilin 3A1 (BTN3A1). In this study, we show that butyrophilin 2A1 (BTN2A1) is required for BTN3A-mediated Vγ9Vδ2 T cell cytotoxicity against cancer cells, and that expression of the BTN2A1/BTN3A1 complex is sufficient to trigger Vγ9Vδ2 TCR activation. Also, BTN2A1 interacts with all isoforms of BTN3A (BTN3A1, BTN3A2, BTN3A3), which appears to be a rate-limiting factor to BTN2A1 export to the plasma membrane. BTN2A1/BTN3A1 interaction is enhanced by pAgs and, strikingly, B30.2 domains of both proteins are required for pAg responsiveness. BTN2A1 expression in cancer cells correlates with bisphosphonate-induced Vγ9Vδ2 T cell cytotoxicity. Vγ9Vδ2 T cell killing of cancer cells is modulated by anti-BTN2A1 monoclonal antibodies (mAbs), whose action relies on the inhibition of BTN2A1 binding to the Vγ9Vδ2TCR. This demonstrates the potential of BTN2A1 as a therapeutic target and adds to the emerging butyrophilin-family cooperation pathway in γδ T cell activation.

Background Vg9Vd2 T constitute the predominant subset among gd T cells in peripheral blood. Their infiltration into malignant tissues is associated with a favorable prognosis. Their anti-tumor activity is triggered by intracellular accumulation of organic phosphoantigens (pAgs) due to tumorigenesis. Recently, BTN2A1 was shown to bind to the Vg9TCR chain allowing immune synapse between cancer and Vg9Vd2T cells, thus initiating the anti-tumoral response. In this study, we generated monoclonal antibodies against BTN2A1 and evaluated their ability to modulate γδT cell cytotoxicity. Methods Anti-BTN2A1 mAbs were generated by mouse immunization. Their effect on Vg9Vd2 T cell degranulation, secretion of IFNγ/TNFα, and target cell killing as depicted by caspase 3/7 cleavage, were tested in co-cultures with Daudi, HL-60 cell lines and primary acute myelocytic leukemia (AML) blasts with or without zoledronate or the anti-BTN3A mAb 20.1. These readouts were measured by flow cytometry. Endometrial cancer spheroids were used to assess the ability of the anti-BTN2A1 antagonistic mAb to inhibit Vg9Vd2 T cell killing of cancers cells. Results We generated 7 anti-BTN2A1 mAbs and tested their effect on Vg9Vd2 T cell degranulation against Daudi cells with or without zoledronate. Six out of 7 anti-BTN2A1 mAbs significantly inhibited basal Vg9Vd2 T cell degranulation against Daudi up to 17-fold, and 5 of them were able to inhibit Vg9Vd2 T cell degranulation against Daudi in presence of zoledronate. Consistently, anti-BTN2A1 mAbs abrogated zoledronate and anti-BTN3A 20.1-induced apoptosis with different efficiencies. The level of apoptosis inhibition after zoledronate and 20.1 treatment were correlated. Anti-BTN2A1 7.48 mAb was the clone with the highest inhibitory potential. Increasing concentrations of 7.48 abrogated not only Vg9Vd2 T cell degranulation (IC50= 0.033±0.0003 µg/mL) but also TNFα (IC50= 0.03±0.006 µg/mL) and IFNγ (IC50= 0.015±0.004 µg/mL) secretion against Daudi cells in presence of pAgs. The ability of anti-BTN2A1 antibodies to inhibit Vg9Vd2 induced tumor cell apoptosis was also shown in 3D endometrial cancer spheroids. In co-cultures of Vg9Vd2 T cells with primary AML blasts, the anti-BTN2A1 7.48 inhibited Vg9Vd2 T cell degranulation as well as TNFα, IFNγ production and killing of AML blasts. Conclusions Antagonist antibodies to BTN2A1 highlighted its critical role in Vg9Vd2 anti-tumor responses. BTN2A1 is involved in Vg9Vd2 T cell anti-tumoral activity and can constitute an interesting therapeutic target for gdT cell response immunomodulation in cancer or immune diseases treatment. Ethics Approval The research was approved by the relevant institutional review boards (ethic committee and ANSM, HEMATO-BIO IPC 2013-015, Ref ANSM 131368B-11, Sponsor Institut Paoli Calmettes N° ID RCB 2013-A01437-38). Consent Informed consent was obtained from all donors in accordance with the 121 Declaration of Helsinki.

The treatment of advanced-stage cutaneous T-cell lymphoma (CTCL) remains an unmet medical need. Mogamulizumab, anti-KIR3DL2, and brentuximab vedotin (BV), an anti-CD30 antibody–drug conjugate (ADC) coupled with monomethyl-auristatin-E (MMAE), provided encouraging results, but new targeted therapies are needed. Inducible T-cell costimulator (ICOS), a T-cell costimulatory receptor, is a promising therapeutic target, not only because it is expressed by malignant T cells in CTCL but also because of its connection with the suppressive activity of regulatory T (Treg) cells. Immunohistochemical analysis revealed that ICOS was widely expressed by malignant cells in skin biopsy specimens from 52 patients with mycosis fungoides and Sézary syndrome (SS), as well as in involved node biopsy specimens from patients with SS. Furthermore, flow cytometry demonstrated its strong expression by circulating tumor cells in all our patients with SS. Percentages of ICOS+ Treg cells were significantly higher in patients with SS than in healthy donors. We then investigated the preclinical efficacy of anti-ICOS ADCs generated by coupling murine anti-ICOS monoclonal antibodies with MMAE and pyrrolobenzodiazepine. In 3 CTCL cell lines (Myla, MJ, and HUT78), we observed a significant dose-dependent decrease in cell viability in the presence of anti-ICOS ADCs. In addition, anti-ICOS-MMAE ADCs had an in vitro and in vivo efficacy superior to BV in a mouse xenograft model (MyLa). Finally, we assessed the efficacy of anti-ICOS ADCs in ICOS+ patient-derived xenografts from patients with SS and angioimmunoblastic T-cell lymphoma. Collectively, our findings provide the preliminary basis for a therapeutic trial.

International audience; No abstract available

Checkpoint inhibitors have become an efficient way to treat cancers. Indeed, anti-CTLA-4, anti-PD1, and anti-PDL-1 antibodies are now used as therapies for cancers. However, while these therapies are very efficient in certain tumors, they remain poorly efficient in others. This might be explained by the immune infiltrate, the expression of target molecules, and the influence of the tumor microenvironment. It is therefore critical to identify checkpoint antigens that represent alternative targets for immunotherapies. PVR-like molecules play regulatory roles in immune cell functions. These proteins are expressed by different cell types and have been shown to be upregulated in various malignancies. PVR and Nectin-2 are expressed by tumor cells as well as myeloid cells, while TIGIT, CD96, and DNAM-1 are expressed on effector lymphoid cells. PVR is able to bind DNAM-1, CD96, and TIGIT, which results in two distinct profiles of effector cell activation. Indeed, while binding to DNAM-1 induces the release of cytokines and cytotoxicity of cytotoxic effector cells, binding TIGIT induces an immunosuppressive and non-cytotoxic profile. PVR is also able to bind CD96, which induces an immunosuppressive response in murine models. Unfortunately, in humans, results remain contradictory, and this interaction might induce the activation or the suppression of the immune response. Similarly, Nectin-2 was shown to bind TIGIT and to induce regulatory profiles in effectors cells such as NK and T cells. Therefore, these data highlight the potential of each of the molecules of the “PVR–TIGIT axis” as a potential target for immune checkpoint therapy. However, many questions remain to be answered to fully understand the mechanisms of this synapse, in particular for human CD96 and Nectin-2, which are still understudied. Here, we review the recent advances in “PVR–TIGIT axis” research and discuss the potential of targeting this axis by checkpoint immunotherapies.

International audience; Introduction: The promotion of antitumor response by targeting co-stimulatory B7 superfamily members has become evident to create a new wave of cancer immunotherapy. Inducible Co-Stimulator (ICOS), which is expressed on activated T cells, gained interest in the translational medicine community.Areas covered: We performed an extensive literature review using the keywords 'ICOS' and 'cancer', and the Clinicaltrials.gov database for early phase clinical trials targeting ICOS. In this review, we highlight the dual role of ICOS in oncogenesis in different malignancies. We summarize the current state of knowledge about ICOS/ICOSL pathway targeting by immunotherapies.Expert opinion: Due to its multifaceted link with anti-tumor immunity, both antagonist and agonist antibodies might be of interest to target the ICOS/ICOSL pathway for tumor treatment. Indeed, ICOS activation might potentiate the effect of an inhibitory checkpoint blockade, while its neutralization could decrease the function of immunosuppressive Tregs and inhibit lymphoid tumor cells expressing Tfh markers.

Background: In a previous study, we reported the strong expression of Inducible T-cell costimulatory (ICOS) by Sézary cells, and presented the excellent preclinical efficacy results of anti-ICOS antibody drug conjugates (ADCs) in both Sézary syndrome (SS) and angioimmunoblastic T-cell lymphoma. Although exerting a potent direct action on the tumor cell, ADCs have the disadvantage of being associated with a cumulative toxicity, related to the conjugated drug. The development of antibody-dependent cell-mediated cytotoxicity/phagocytosis (ADCC/ADCP)-inducing anti-ICOS monoclonal antibodies (mAbs) is therefore of great importance to ensure long-term maintenance therapy. Methods: We first determined which anti-ICOS clone was the best candidate to induce an ADCC effect on ICOS + cell lines (MyLa, MJ and HUT78), using Mouse FcγRIII ADCC Bioassay (Promega®). The selected mAb was then chimerized and afucosylated (GlymaxX® technology, Evitria®). Secondly, we evaluated in vitro ADCC and ADCP effect of the chimeric anti-ICOS mAb against ICOS + CTCL cell lines, compared to both positive controls (mogamulizumab (moga) and alemtuzumab) and negative controls (IgG1 isotype control, and rituximab). To perform ADCC experiments, we co-incubated target cells with mAbs and allogenic NK lymphocytes from healthy volunteers (HV). Cellular apoptosis was measured by flow cytometry using the Caspase 3/7 assay (Promega®). For ADCP, monocytes were sorted from HV blood samples and treated with M-CSF. Target cells were labeled with PKH67 (Sigma-Aldrich®) and after co-incubation, CD14 +CD11b +PKH67 + monocytes were analyzed by flow cytometry. Finally, we verified the ADCC/ADCP potency of anti-ICOS mAbs on primary Sézary cells isolated from 16 moga-naïve SS patients, and 6 patients who had developed resistance to moga. We also questioned whether anti-ICOS mAbs were able to promote the autologous apoptosis of Sézary cells and T regulatory cells (Tregs) when directly incubated with peripheral blood mononuclear cells (PBMCs) from patients with SS. Results: Among 7 different anti-ICOS clones, 314.8, 92.17 and 293.1 clones had the higher ADCC activity against MyLa, MJ and HUT78. Of these 3 clones, 314.8 had the best affinity to the receptor, and the best ability to inhibit binding between ICOS receptor and a recombinant ICOS ligand protein. Anti-ICOS 314.8 mAb was then chosen to be chimerized and glyco-engineered. ICOS and CCR4 were strong and similarly expressed on MyLa and MJ. HUT78 had only mild expression of ICOS and CD52. Anti-ICOS mediated potent ADCC of cell lines (respectively 39.1±5% and 52.6±2.4% for MyLa and MJ cells), without significant difference when compared to mogamulizumab. In HUT78 cells, anti-ICOS induced a specific apoptosis of 35.7±5% versus 15.6±5.6% with alemtuzumab (p=0.02; CI95%: 4.1-36.1). Moreover, phagocytosis induced by anti-ICOS was significantly increased than that induced by negative controls. On MyLa cells, anti-ICOS had a greater phagocytosis activity than moga (59.4±5.2% vs 39.4±5.1%, p=0.031). Expression of ICOS by circulating tumor cells was found in all the 16 moga-naïve patients. The expression was strong, as 61±6% of tumor cells expressed ICOS vs 20±8% of non-tumoral CD4 + cells. CCR4 was more expressed than ICOS on both Sézary cells and non-tumoral CD4 + cells (91±6%, and 44±9% respectively). Anti-ICOS induced the apoptosis of 57.1±4.7% Sézary cells, compared to 16.9±2.2% with rituximab (p Ex vivo, anti-ICOS allowed 39.4±19.9% and 70.1±20.1% lysis of Sézary cells and Tregs respectively, with no difference with moga and alemtuzumab. However, the depletion of non-tumoral CD4 + and total PBMCs was significantly lower with anti-ICOS mAbs than with moga and alemtuzumab. Discussion: In a recent study, we showed that Treg cells of patients with SS have a high expression of ICOS. Here, we demonstrate that anti-ICOS mAbs induce Tregs depletion, which may improve immune profiles and emphasize Sézary cells killing. Our data suggest robust anti-tumor activity of anti-ICOS mAbs in SS, and xenograft experiments are underway to confirm these findings. Disclosures Lopez: Emergence Therapeutics: Current holder of individual stocks in a privately-held company. Bagot: Takeda: Membership on an entity's Board of Directors or advisory committees. Olive: Alderaan Biotechnology: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; ImCheck Therapeutics: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Emergence Therapeutics: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees.

Introduction Vγ9Vδ2 T cells are new promising cytotoxic effectors in hematological malignancies. In acute myeloid leukemia and in non-Hodgkin lymphomas, Vγ9Vδ2 T cells-based immunotherapy has shown encouraging results both in preclinical models and in early phase clinical trials. Acute lymphoblastic leukemia (ALL) includes very heterogeneous clinico-biological entities, for which recent immunotherapy approaches are currently being developed. Nevertheless, global prognosis of ALL patients still be poor with a 5 years-overall survival of less than 40% and therefore, treatments need to be improved. Very few data are currently available on susceptibility of ALL blasts to Vγ9Vδ2 T cell cytotoxic activity. Vγ9Vδ2 T cells are activated by phosphoantigens bound to BTN3A1 on target cells. BTN3A molecules are targeted at clinical level, with the ICT01 agonist monoclonal antibody (mAb), that is currently tested in a multicentric phase ½ study (EVICTION study). Biology of Vγ9Vδ2 T cells has recently undergone a new paradigm with the identification of BTN2A1 as the direct ligand for Vγ9 chain of γδ TCR. BTN2A1 is mandatory for Vγ9Vδ2 T cell activation but its precise role in modulating functions of Vγ9Vδ2 T cells remains unknown. Here, we show that allogenic and autologous Vγ9Vδ2 T cells exert cytolytic functions against ALL cell lines and primary ALL blasts, and we report that Vγ9Vδ2 T cell cytotoxic activity is enhanced after treatment with a unique agonist mAb targeting BTN2A1. Material and methods 5 ALL cell lines (697, RS4;11, NALM-6, HPB-ALL, SUP-T1) and PBMC from 11 adults ALL patients at diagnosis (B-ALL, T-ALL and Ph+ ALL) were tested in functional assays. We evaluated apoptosis of ALL cell lines and of primary ALL blasts after coculture with allogenic Vγ9Vδ2 T cells. ALL samples were also tested for their expansion capacities and a degranulation assay was performed at D14. We assessed in parallel relative quantification of the level expression of BTN2A1 (ICT0302 and 7.48 epitopes), and BTN3A (20.1 and 108.5 epitopes) on surface of ALL blasts. DAUDI-BTN2AKO+2A1 and HEK293-BTN2AKO+2A1 cells were used in binding assays, and modulation of TCR binding was assessed using recombinant tetramerized Vγ9Vδ2 TCR. Results We showed that Vγ9Vδ2 T cells exert spontaneous cytotoxicity against ALL cell lines and primary ALL blasts with a heterogeneous susceptibility depending on the target. We demonstrated that anti-BTN2A1 ICT0302 agonist mAb significantly enhanced Vγ9Vδ2 T cells mediated apoptosis in comparison to control condition, even for the less spontaneously susceptible cells. We confirmed these observations with degranulation of autologous Vγ9Vδ2 T cells expanded from 5 ALL patients at diagnosis that was increased after treatment with anti-BTN2A1 ICT0302 agonist mAb. BTN3A and BTN2A1 were detected on surface of ALL blasts, and BTN3A 108.5 was the most expressed epitope. Interestingly, we observed that anti-BTN2A1 ICT0302 strongly increased binding of a recombinant Vγ9Vδ2 TCR to target cells using with HEK293 and DAUDI cells. Discussion Our results highlighted that Vγ9Vδ2 T cells exert cytolytic functions against ALL cells, both in allogenic and autologous setting and demonstrated that BTN2A1 targeting with our unique agonist mAb could potentiate effector activities of Vγ9Vδ2 T cells against ALL blasts. These results indicate that the sensitization of leukemic cells can be induced by activation BTN3A as well as BTN2A1 mAbs. These data bring novel understanding on the biology of BTN2A1 on leukemic cells and our ability to enhance both binding and function. These findings could be of great interest for the design of innovative Vγ9Vδ2 T cells-based immunotherapy strategies for treating ALL that could be extended to other cancer types. Disclosures De Gassart: ImCheck Therapeutics: Current Employment, Current holder of individual stocks in a privately-held company. Vey: Amgen: Honoraria; BMS: Honoraria; BIOKINESIS: Consultancy, Research Funding; NOVARTIS: Consultancy, Honoraria, Research Funding; SERVIER: Consultancy; JAZZ PHARMACEUTICALS: Honoraria; JANSSEN: Consultancy. Cano: ImCheck Therapeutics: Current Employment, Current holder of individual stocks in a privately-held company. Olive: Emergence Therapeutics: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Alderaan Biotechnology: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; ImCheck Therapeutics: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees. OffLabel Disclosure: Anti-BTN2A1 ICT0302 is a murine agonist monoclonal antibody targeting BTN2A1 whose aim is to increase Vgamma9Vdelta2 T cells functions.

Cytokines produced by dendritic cells (DCs) can largely determine the direction of immunity. Transcriptional analysis revealed that besides IL-15, IL-32 was the only other cytokine expressed by human Langerhans cells. IL-32 is a human cytokine that exists in four main isoforms. Currently, little is known about the regulation and function of the various IL-32 isoforms. In this study, we found that IL-15 is a potent inducer of IL-32α in DCs. Because IL-15 promotes NK cell activation, we investigated the interplay between IL-32 and IL-15 and their role in NK cell activity. We show that IL-32α acts on NK cells to inhibit IL-15–mediated STAT5 phosphorylation and to suppress their IL-15–induced effector molecule expression and cytolytic capacity. IL-32α also acted on DCs by downregulating IL-15–induced IL-18 production, an important cytokine in NK cell activity. Blocking IL-32α during DC:NK cell coculture enhanced NK cell effector molecule expression as well as their cytolytic capacity. Taken together, our findings suggest a feedback inhibition of IL-15–mediated NK cell activity by IL-32α.

For 40 years, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been widely used in proteomics and biochemistry. It has been demonstrated in the last decade that MALDI-TOF MS can be used routinely to identify and classify numerous bacterial species or subspecies. We applied MALDI-TOF MS directly to intact mammalian cells, and we found that this method is valuable to identify human circulating cells and cells involved in the immune response including macrophages. We then stimulated human macrophages with cytokines, bacterial products, and a variety of bacteria. We found that MALDI-TOF MS discriminated unstimulated and stimulated macrophages and also detected multifaceted activation of macrophages. We conclude that whole-cell MALDI-TOF MS is an accurate method to identify various cell types and to detect subtle modifications in cell activity and therefore it can be beneficial in clinical practices for a rapid patient classification based on their immune profile.