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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.

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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.

Background Butyrophillin-3A (BTN3A) three isoforms (3A1/3A2/3A3) are widely expressed on a variety of tumors.1 BTN3A1 plays a key role in phosphoantigen activation of Vg9Vd2 T-cells, key mediators of innate and adaptive anti-tumor immunity.2 Vg9Vd2 T-cell infiltration into tumor tissues is associated with a positive prognosis across multiple cancers,3 which makes BTN3A an interesting target for enhancing anti-tumor immunity. ImCheck Therapeutics is developing ICT01, an anti-BTN3A mAb that specifically activates Vg9Vd2 T-cells. ICT01 is currently in an international, multi-center Phase 1/2a clinical trial (NCT04243499, EVICTION Study). The level of BTN3A expression required for a clinical response to ICT01 is not known. Therefore, we developed novel immunohistochemistry (IHC) methods to enable a precision-medicine based approach to target population selection for dose escalation and potentially guiding patient selection in the expansion cohorts of the ongoing EVICTION study. Methods A panBTN3A IHC staining that recognizes the three isoforms was developed on Fresh frozen (FF) tissues, while BTN3A2- and BTN3A3-specific IHC methods were developed on formalin-fixed paraffin embedded (FFPE) tissues. BTN3A1-specific staining is still under development. Transfected knock-out/knock-in cell lines and positive tissues were used to assess antibody specificity. BTN3A expression was then analyzed on both normal and associated tumor tissue using tissue microarrays (TMA) and selected frozen blocks from tumor biopsies. FACS analyses were also performed on dissociated lung and pancreatic cancer biopsies to determine BTN3A (3 isoforms) membrane expression on tumor-infiltrating immune cells and cancer/stromal cells. Results In normal tissues, BTN3A2 and BTN3A3 specific IHC signals were granular cytoplasmic in epithelial cells, with positive mononuclear and endothelial cells. Higher expression in lung, colon, and small intestine tissues was observed. Regarding panBTN3A expression, inter-indication and inter-patient heterogeneity was observed among head and neck, lung, melanoma, bladder, colon, pancreas, breast, and prostate cancer tissues, with both cytoplasmic and membranous localizations. The major finding was higher expression of BTN3A2 on malignant cells in melanoma, lung, colon, and prostate cancers, as compared to normal tissue. Finally, FACS analyses of lung and pancreatic cancer tissues revealed stronger expression of all BTN3A isoforms at the cell surface of infiltrated immune cells compared to its expression on stromal cells. Conclusions These validated IHC methods supported the selection of cancer indications for the EVICTION trial and will potentially help identify specific tumor subtypes and patients that will most likely benefit from ICT01 treatment. References Compte E, Pontarotti P, Collette Y, Lopez M, Olive D. Frontline: characterization of BT3 molecules belonging to the B7 family expressed on immune cells. Eur J Immunol 2004;34:2089–99. Juan-Luis Blazquez, Audrey Benyamine, Christine Pasero, and Daniel Olive. New Insights into the Regulation of ?d T Cells by BTN3A and Other BTN/BTNL in Tumor Immunity. Front Immunol 2018;9:1601. Andrew J. Gentles, Aaron M. Newman, Chih Long Liu, Scott V. Bratman, Weiguo Feng, Dongkyoon Kim, Viswam S. Nair, Yue Xu, Amanda Khuong, Chuong D. Hoang, Maximilian Diehn, Robert B. West, Sylvia K. Plevritis, Ash A. Alizadeh. The prognostic landscape of genes and infiltrating immune cells across human cancers. Nat Med 2015 Aug; 21(8):938–945.

Natural killer (NK) cells are major anti-leukemic 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 present 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 evidenced 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, with decreased overall survival (HR=0.13; P=.0002) and event-free survival (HR=0.33; P=.018), and retained statistical significance in multivariate analysis. Pseudo-time 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.SignificanceThis work provides the first report of accumulation of unconventional CD56-CD16+ NK cells in non-virally induced malignancies. Pseudotime analysis highlights a bifurcation point occurring during the course of NK cell maturation, providing elements regarding the possible origin of CD56-CD16+ NK cells. Increased frequency of CD56-CD16+ NK cells is associated with adverse clinical outcome in AML and might contribute, as well as other maturation defects, to a defective control of AML progression. Overall, accumulation of CD56-CD16+ NK cells could be an important feature of immune escape from innate immunity.Graphical abstractKey pointsA disruption in the maturation process of NK cells leads to accumulation of unconventional CD56- CD16+ NK cells in patients with AMLHigh frequency of CD56-CD16+ NK cells is associated with adverse clinical outcome

Cytogenetics and European Leukemia Net (ELN) genetic classification predict patients at increased risk of relapse in acute myeloid leukemia (AML) except in the intermediate risk group for which further prognostic determinants are required. We have previously shown that Natural Killer (NK) cell defects in AML are predictors of poor overall survival (OS). This study aimins at validating NKp30, a receptor that mediates NK activation, as a prognostic biomarker for AML patients with intermediate prognosis. NKp30 expression was prospectively assessed at diagnosis on NK cells from peripheral blood by flow cytometry (N = 201 patients). Clinical outcome was evaluated with regard to NKp30 status. In patients with intermediate cytogenetic (N = 162), NKp30high phenotype at diagnosis was predictive of better OS (HR = 0.26; 95%CI = [0.14-0.50]; P < 0.0001) and relapse-free survival (RFS) (HR = 0.21; 95%CI = [0.08-0.52]; P = 0.0007). In patients with intermediate ELN (N = 116), NKp30high phenotype at diagnosis was predictive of better OS (HR = 0.33; 95%CI = [0.16–0.67]; P = 0.0019) and RFS (HR = 0.24; 95%CI = [0.08-0.67]; P = 0.0058). In multivariate analysis, high NKp30 expression independently predicted improved OS (HR = 0.56, P = 0.046) and RFS (HR = 0.37, P = 0.048). Consistently, cumulative incidence of relapse (CIR) was lower in patients with high NKp30 expression (HR = 0.37, P = 0.026). In conclusion, we propose NKp30 status as a simple and early prognostic biomarker that identifies intermediate-risk patients with poor prognosis who otherwise may not be identified with existing risk stratification systems.

Introduction La maladie associee aux IgG4 est une pathologie fibro-inflammatoire d’etiologie inconnue. Elle est caracterisee par des lesions histologiques typiques, retrouvees dans les differents tissus atteints. Celles-ci temoignent d’une activation lymphocytaire T et B oligoclonale suggerant une stimulation antigenique chronique. Des modifications des populations T helper, T follicular helper et une augmentation des plasmablastes dans le sang ont ete rapportes. Il n’existe pas actuellement de marqueur biologique fiable pour evaluer l’activite de la maladie et le risque de rechute. Nous avons analyse de facon systematique le compartiment lymphocytaire B de ces patients. Materiels et methodes Nous avons analyse prospectivement par cytometrie de flux sur cellules mononuclees du sang circulant les sous populations lymphocytaires B : totales, naives, transitionnelles, memoires commutees, CD21low et plasmablastes de 36 patients presentant une maladie associee aux IgG4 (MAG4) avant traitement. Les donnees ont ete comparees a un groupe controle sains (n = 39) et malades (n = 20, Gougerot-Sjogren). Pour certains patients les valeurs avant et apres traitement ont pu etre comparees. Resultats L’âge moyen des 36 patients est de 62 ans, 95 % sont des hommes. Les formes sont localisees (40 %) ou systemiques (60 %). Les atteintes les plus frequentes sont ganglionnaires (60 %), pancreatiques (42 %), salivaires (31 %), retroperitoneales (25 %) ou biliaire (22 %). Le taux d’IgG4 serique median est de 4 g/L (IQR 1,38–14,22). Compare aux controles sains le % de lymphocytes B CD19+ ou CD20+ etait identique. On observe une elevation significative des B CD27-IgD + IgM+ naives (69,90 versus 57,10) et des plasmablastes (1063 versus 252 cellules/mm3), une diminution des B CD27 + IgD-IgM- memoires commutees (12,45 versus 15,0) et une elevation du % de la population B CD21 low (11,30 versus 6,0). Le % et le taux de B CD21low est plus eleve chez les patients presentant des rechutes apres traitement. Discussion L’elevation des plasmablastes n’est pas la seule modification des lymphocytes B circulants au cours de la MAG4. Nous rapportons des modifications quantitatives des populations B naives, memoires et CD21low au cours de la maladie. Les cellules B CD21low n’ont pas de mutations somatiques, expriment une IgM de surface. L’elevation de la population CD21low a ete rapportee au cours de l’infection par le VIH, le VHC, au cours du Lupus et dans le deficit immunitaire commun variable (DICV). Dans le cas du DICV sa presence correle avec l’existence d’une splenomegalie et/ou d’une granulomatose associee. Leur expansion serait liee a une stimulation antigenique chronique. Il semblerait exister une correlation entre leur taux avant traitement et le risque de rechute au cours de la MAG4. Conclusion La MAG4 est associee a des modifications qualitatives des sous population B circulantes principalement une modification de la balance B naives/memoires et une elevation des CD21low.Le role de ces changements dans la physiopathologie et leur utilite dans la caracterisation clinique et pronostique de patients necessite d’etre precisee.

Introduction Community-acquired meningitis is a monomicrobial infection caused by either viruses or bacteria in the vast majority of patients. We report here one exceptional case of a patient with mixed bacterial meningitis due to Streptococcus pneumoniae and Streptococcus pyogenes. Case presentation We report the case of a 68-year-old immunocompromised Caucasian man suffering from otitis and then meningitis caused by Streptococcus pneumoniae and Streptococcus pyogenes. Bacteria were undistinguishable by direct microscopic examination of the cerebrospinal fluid. He responded well to treatment with cefotaxime and dexamethasone, with no sequelae observed at the 4-month follow-up. Conclusions This first reported case of mixed S. pneumoniae and S. pyogenes meningitis illustrates the life-threatening consequences of barotrauma in immunocompromised patients suffering from otorhinolaryngeal infections.