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Melanoma is responsible for 90% of skin cancer-related deaths. Major therapeutic advances have led to a considerable improvement in the prognosis of patients, with the development of targeted therapies (BRAF or MEK inhibitors) and immunotherapy (anti-CTLA-4 or -PD-1 antibodies). However, the tumor constitutes an immunosuppressive microenvironment that prevents the therapeutic efficacy and/or promotes the development of secondary resistances. CD160 is an activating NK-cell receptor initially described as delineating the NK and CD8+T-cell cytotoxic populations. Three forms of CD160 have been described: (1) the GPI isoform, constitutively expressed and involved in the initiation of NK-cells' cytotoxic activity, (2) the transmembrane isoform, neo-synthesized upon cell activation, allowing the amplification of NK cells' cytotoxic functions and (3) the soluble form, generated after cleavage of the GPI isoform, which presents an immuno-suppressive activity. By performing immunohistochemistry analyses, we observed a strong expression of CD160 at the primary cutaneous tumor site of melanoma patients. We further demonstrated that melanoma cells express CD160-GPI isoform and constitutively release the soluble form (sCD160) into the tumor environment. sCD160 was shown to inhibit the cytotoxic activity of NK-cells towards their target cells. In addition, it was found in the serum of melanoma patients and associated with increased tumor dissemination. Altogether these results support a role for sCD160 in the mechanisms leading to the inhibition of anti-tumor response and immune surveillance in melanoma.

T-cell lymphomas are a heterogeneous group of malignancies involving T lymphocytes and generally characterized by a poor prognosis. Among them, cutaneous T-cell lymphomas (CTCL) involve primarily the skin. The most frequent CTCL are mycosis fungoides and Sézary syndrome (SS) (1). There is no cure for advanced-stage CTCL, the remissions are generally rare and short, and the overall survival is around 5 years with an impaired quality of life. Thus, the development of new treatments inducing longstanding responses in CTCL is a major unmet need (2). Because Sézary cells express several Treg markers as PD-1, ICOS, CD39 or CCR8 (3), we analyzed the expression of CD51, the αV integrin, which is expressed with the β8 integrin on Treg cells (4). CD51 is involved in the production of active form of TGF-β, a cytokine with immunosuppressive properties that promotes tumor escape. Thus, targeting the αVβ8 integrin with a specific antibody has been shown to inhibit TGF-β activation while not inhibiting cell adhesion (5). Our results revealed that CD51 is significantly overexpressed in circulating lymphocytes from SS patients as compared to healthy individuals. Interestingly, CD51 is not associated with the β8 integrin but with CD29 (β1 integrin), and CD61 (β3 integrin) in some patients.CD51/CD29 and/or CD51/CD61 heterodimers appear as potential therapeutic targets in T-cell lymphoma, which may be associated with the production of active TGF-β. Targeting these heterodimers with specific antibodies to prevent the release of active TGF-β could improve patient’s antitumor immune response. They could also represent depleting targets through NK or CAR-T cells activities as shown in diffuse intrinsic pontine glioma and glioblastoma for CD51/CD61 heterodimer (6). 1. Dobos G, et al. Epidemiology of Cutaneous T-Cell Lymphomas: A Systematic Review and Meta-Analysis of 16,953 Patients. Cancers. 11 oct 2020;12(10).2. Scarisbrick JJ, et al. The changing therapeutic landscape, burden of disease, and unmet needs in patients with cutaneous T-cell lymphoma. Br J Haematol. févr 2021;192(4).3. Giustiniani J, et al. CCR8 is a new therapeutic target in cutaneous T-cell lymphomas. Blood Adv. 2022 Feb 24.4. Stockis J, et al. Blocking immunosuppression by human Tregs in vivo with antibodies targeting integrin αVβ8. Proc Natl Acad Sci U S A 2017 Nov 21;114(47).5. Tsui P, et al. The TGF-β inhibitory activity of antibody 37E1B5 depends on its H-CDR2 glycan. mAbs. janv 2017;9(1):104-13.6. Cobb D, de Rossi J, Liu L, An E, Lee D. Targeting of the alphav beta3 integrin complex by CAR-T cells leads to rapid regression of diffuse intrinsic pontine glioma and glioblastoma J Immunother Cancer 2022. Citation Format: Jérôme Giustiniani, Anne Marie-Cardine, Sadjia Belkhelouat, Martine Bagot, Armand Bensussan, Nicolas Ortonne, Adèle De Masson. Targeting the CD51/TGF-beta pathway: a potential innovative cancer immunotherapy in T-cell lymphomas. [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 3644.

Background: Due to their basal stress phenotype associated to transformation, cancer cells are addicted to non-mutated, non-oncogenic proteins that do not bear such vital functions in normal cells, a phenomenon referred as Non-Oncogenic Addiction (NOA). Targeting these NOAs could therefore induce selective killing of cancer cells, opening several therapeutic opportunities. Recent data suggest that the stress-related scaffold protein AAC-11 (anti-apoptosis clone 11, also known as Api5) is critically involved in cancer cells resistance to chemotherapies, metastatic potential and escape from the immune system. Methods: We have developed inactivating peptides based on the fusion of a cell penetrating sequence and portions of the leucine-zipper domain of AAC-11, which functions as a protein-protein interaction module. These peptides induce cancer cells death, through the inhibition of protein-protein interactions between AAC-11 and its partners, while sparing normal cells. We now describe further characterization of our lead peptide, JRT39, which contains residues 377 to 379 of AAC-11 linked to the cell-penetrating peptide “penetratin”. Results: In vitro, JRT39 causes cell death in a wide spectrum of cancer cell lines with IC50 ranging from 5 µM to 30 µM depending on tumor cell type. In particular, JRT39 showed selective efficacy towards primary cells from Acute Myeloid Leukemia (AML), Acute Lymphocytic Leukemia (ALL) or Sézary syndrome (SS) patients, while sparing normal hematopoietic cells, with an IC50 of 5-15 µM. Mechanistically, JRT39 induces membranolysis of cancer cells through binding to p21-activated kinase 1 (PAK1) in AML or SS cells plasma membrane, where PAK1 is overexpressed. In addition, JRT39 exerted potent anti-tumor activity in vivo in disseminated or subcutaneous AML, APL (Acute Promyelocytic Leukemia) and SS-patient derived preclinical murine models. Preliminary pharmacokinetic studies revealed that JRT39 is stable in human serum and has a plasma half-life of ~1.5-2.5 hours after intravenous (IV) administration to dogs or cynomolgus monkeys, with concentration-time data fitting 2-compartment model. Finally, JRT39 was well tolerated at 5 and 10 mg/kg after single or repeated (daily) IV injections. Conclusions: Combined, our preclinical data confirm that interfering with AAC-11-related survival pathways is a promising novel anticancer strategy and support the development of JRT39 for the treatment of cancer. Citation Format: Louise Jeammet, Emile Adicéam, Justine Habault, Anna Kaci, Jeannig Berrou, Mélanie Dupont, Nicolas Thonnart, Ewa Pasquereau-Kotula, Anne Marie-Cardine, Armand Bensussan, Marika Pla, Hervé Dombret, Claude Gardin, Martine Bagot, Jean-Christophe Rain, Hélène Sicard, Jérôme Tiollier, Thorsten Braun, Jean-Luc Poyet. AAC-11 survival pathways as therapeutic target in cancer: AAC-11 leucine-zipper domain derived peptides exert potent antitumor effects and exhibit favorable stability, pharmacokinetic and toxicology profiles [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 464.

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.

TIGIT is an immune checkpoint inhibitor expressed by effector CD4 þ and CD8 þ T cells, NK cells, and regulatory T cells (Tregs). Inhibition of TIGIT-ligand binding using antagonistic anti-TIGIT mAbs has shown in vitro potential to restore T-cell function and therapeutic efficacy in murine tumor models when combined with an anti–PD(L)-1 antibody. In the current work, we demonstrate broader TIGIT expression than previously reported in healthy donors and patients with cancer with expression on gd T cells, particularly in CMV-seropositive donors, and on tumor cells from hematologic malignancies. Quantification of TIGIT density revealed tumor-infiltrating Tregs as the population expressing the highest receptor density. Consequently, the therapeutic potential of anti-TIGIT mAbs might be wider than the previously described anti–PD(L)-1-like restoration of ab T-cell function. CD155 also mediated inhibition of gd T cells, an immune population not previously described to be sensitive to TIGIT inhibition, which could be fully prevented via use of an antagonistic anti-TIGIT mAb (EOS-448). In PBMCs from patients with cancer, as well as in tumor-infiltrating lymphocytes from mice, the higher TIGIT expression in Tregs correlated with strong antibody-dependent killing and preferential depletion of this highly immunosuppressive population. Accordingly, the ADCC/ADCP–enabling format of the anti-TIGIT mAb had superior antitumor activity, which was dependent upon Fcg receptor engagement. In addition, the anti-TIGIT mAb was able to induce direct killing of TIGIT-expressing tumor cells both in human patient material and in animal models, providing strong rationale for therapeutic intervention in hematologic malignancies. These findings reveal multiple therapeutic opportunities for anti-TIGIT mAbs in cancer therapeutics.

International audience; Sézary syndrome is an aggressive form of cutaneous T-cell lymphoma characterized by the presence of a malignant CD4+ T-cell clone in both blood and skin. Its pathophysiology is still poorly understood, and the development of targeted therapies is hampered by the absence of specific target proteins. AAC-11 plays important roles in cancer cell progression and survival and thus has been considered as an anticancer therapeutic target. In this study, we show that a peptide called RT39, comprising a portion of AAC-11‒binding site to its protein partners coupled to the penetratin sequence, induces the specific elimination of the malignant T-cell clone both ex vivo on the circulating cells of patients with Sézary syndrome and in vivo in a subcutaneous xenograft mouse model. RT39 acts by direct binding to PAK1 that is overexpressed, located in the plasma membrane, and constitutively activated in Sézary cells, resulting in their selective depletion by membranolysis. Along with the absence of toxicity, our preclinical efficacy evidence suggests that RT39 might represent a promising alternative therapeutic tool for Sézary syndrome because it spares the nonmalignant immune cells and, contrary to antibody-based immunotherapies, does not require the mobilization of the cellular immunity that shows heavy deficiencies at advanced stages of the disease.

Cutaneous T-cell lymphomas (CTCLs) are rare malignancies involving primarily the skin. Responses to treatment are usually short-lived in advanced CTCL. The determinants of long-term CTCL control are unclear. Mogamulizumab, an anti-human CCR4 antibody that acts by antibody-dependent cell cytotoxicity against CCR4+ CTCL tumor cells and peripheral memory blood regulatory T cells, has been associated with long-lasting remissions and immune adverse events. Here, we reported skin rashes in 32% of 44 patients with CTCL treated with mogamulizumab, associated with significantly higher overall survival (hazard ratio, 0.16; 0.04-0.73; P = .01). Rash occurred in patients with Sézary syndrome and was associated with longer time to progression. These rashes were characterized by a CD163+ granulomatous and/or CD8+ lichenoid skin infiltrate. High-throughput sequencing analysis of T-cell receptor β genes in skin and blood flow cytometry confirmed the depletion of CTCL tumor cells, as well as the recruitment of new reactive T-cell clones in skin at the time of skin rash. CXCL9 and CXCL11, two macrophage-derived chemokines that recruit CXCR3+ T cells to skin, were overexpressed in skin rashes. A higher frequency of TIGIT+ and PD1+ exhausted reactive blood T cells was observed at baseline in patients with rash, and this frequency decreased with mogamulizumab treatment. These data are consistent with mogamulizumab-induced long-term immune CTCL control by activation of the macrophage and T-cell responses in patients with rash.

Summary Background IPH4102 is a first-in-class monoclonal antibody targeting KIR3DL2, a cell surface protein that is expressed in cutaneous T-cell lymphoma, and predominantly in its leukaemic form, Sezary syndrome. We aimed to assess the safety and activity of IPH4102 in cutaneous T-cell lymphoma. Methods We did an international, first-in-human, open-label, phase 1 clinical trial with dose-escalation and cohort-expansion parts in five academic hospitals in the USA, France, the UK, and the Netherlands. Eligible patients had histologically confirmed relapsed or refractory primary cutaneous T-cell lymphoma, an Eastern Cooperative Oncology group performance score of 2 or less, were aged 18 years or older, and had received at least two previous systemic therapies. Ten dose levels of IPH4102, administered as an intravenous infusion, ranging from 0·0001 mg/kg to 10 mg/kg, were assessed using an accelerated 3 + 3 design. The primary endpoint was the occurrence of dose-limiting toxicities during the first 2 weeks of treatment, defined as toxicity grade 3 or worse lasting for 8 or more days, except for lymphopenia. Global overall response by cutaneous T-cell lymphoma subtype was a secondary endpoint. Safety and activity analyses were done in the per-protocol population. The study is ongoing and recruitment is complete. This trial is registered with ClinicalTrials.gov , number NCT02593045 . Findings Between Nov 4, 2015, and Nov 20, 2017, 44 patients were enrolled. 35 (80%) patients had Sezary syndrome, eight (18%) had mycosis fungoides, and one (2%) had primary cutaneous T-cell lymphoma, not otherwise specified. In the dose-escalation part, no dose limiting toxicity was reported and the trial's safety committee recommended a flat dose of 750 mg for the cohort-expansion, corresponding to the maximum administered dose. The most common adverse events were peripheral oedema (12 [27%] of 44 patients) and fatigue (nine [20%]), all of which were grade 1–2. Lymphopenia was the most common grade 3 or worse adverse event (three [7%]). One patient developed possibly treatment-related fulminant hepatitis 6 weeks after IPH4102 discontinuation and subsequently died. However, the patient had evidence of human herpes virus-6B infection. Median follow-up was 14·1 months (IQR 11·3–20·5). A confirmed global overall response was achieved in 16 (36·4% [95% CI 23·8–51·1]) of 44 patients, and of those, 15 responses were observed in 35 patients with Sezary syndrome (43% [28·0–59·1]). Interpretation IPH4102 is safe and shows encouraging clinical activity in patients with relapsed or refractory cutaneous T-cell lymphoma, particularly those with Sezary syndrome. If confirmed in future trials, IPH4102 could become a novel treatment option for these patients. A multi-cohort, phase 2 trial (TELLOMAK) is underway to confirm the activity in patients with Sezary syndrome and explore the role of IPH4102 in other subtypes of T-cell lymphomas that express KIR3DL2. Funding Innate Pharma

Sézary syndrome is an aggressive form of cutaneous T-cell lymphoma characterized by the presence of a malignant CD4

Introduction Le syndrome de Sezary (SS) est un lymphome T cutane rare de mauvais pronostic caracterise par une phase leucemique et rattache au mycosis fongoide (MF) dans la classification internationale. La transformation en grandes cellules (LCT) est un facteur pronostique independant defavorable bien decrit dans le MF. Cependant, la LCT n’a jamais ete etudiee specifiquement sur une large cohorte de patients avec SS. L’objectif principal de notre etude est de decrire la frequence et la valeur pronostique de la LCT dans le SS. Materiel et methodes Nous avons collecte retrospectivement les donnees cliniques, histologiques, biologiques et cytometriques au diagnostic et au cours du suivi de tous les patients ayant un syndrome de Sezary diagnostiques entre 1998 et 2020 selon les criteres OMS/EORTC et suivis a l’hopital Saint-Louis (AP–HP, PARIS). Chaque biopsie cutanee a ete analysee independamment par deux pathologistes puis scannee et analysee numeriquement (Innate Pharma, Marseille). Resultats Un total de 117 patients ont ete inclus dans l’etude. Le suivi median etait de 41 mois [IQR : 1–81]. La LCT diagnostiquee sur biopsie cutanee etait observee chez 6 % (6/100) et 20% (18/91) des patients, respectivement au diagnostic et au cours du suivi. L’etude de la reproductibilite inter-observateur pour le diagnostic de la LCT montrait un coefficient Kappa de Cohen a 0,88 (IC95[0,78–0,98]). L’analyse d’image sur lames scannees (n = 190) retrouvait une surface moyenne des 25 % des cellules les plus grandes egale a 33 μm2 [SD = 4,7] sur les biopsies LCT+ vs 26 μm2 [SD = 3,2] sur les biopsies LCT− ; p 60 ans, le taux de LDH eleve, le taux de cellules circulantes CD3 + CD4 + CD26− ≥ 10 000/mm3 et une LCT au diagnostic etaient associes independamment a une diminution de la survie globale. Discussion La LCT dans le syndrome de Sezary est un facteur pronostique defavorable, independant de la taille des cellules tumorales circulantes, dont l’evaluation histologique est fiable, en utilisant les criteres definis pour le MF, et recommandee au cours du suivi. Notre etude comparant des donnees cliniques, histologiques, biologiques et cytometriques est, a notre connaissance, la premiere decrivant la frequence et la signification pronostique de la LCT dans une large cohorte homogene de SS.

Sezary syndrome (SS) is a rare, aggressive leukemic form of cutaneous T-cell lymphoma characterized by generalized erythroderma, lymphadenopathy and circulating malignant CD4+ T cells (Sezary cells). Sezary syndrome evolves very quickly, patients are severely immunocompromised and their quality of life is drastically impaired. SS prognosis remains dismal despite recent therapeutic advances. AAC-11 (Anti-Apoptosis Clone 11, also known as Api5) is a survival scaffold protein that is widely expressed in cancer cells. Recent data indicate that AAC-11 might to be one of the central molecules involved in cancer cell progression and survival. Therefore, its inactivation might constitute an attractive approach for developing novel cancer therapeutics. AAC-11 contains a heptad leucine repeat domain that constitutes a protein-protein interaction motif, allowing its interaction with several apoptosis regulatory proteins. Loss-of-function mutations of the heptad leucine repeat motif are responsible for a defect in the interaction with its binding partners and associated with an abolition of the survival properties of AAC-11. We have recently described cell penetrating peptides based on the fusion of penetratin and a portion of the heptad leucine repeat region of AAC-11 that can induce cancer cells death. This effect is not observed in normal cells, suggesting high therapeutic indices. Here we evaluated the anti-tumor effect of a novel AAC-11-derived peptide, termed RT39, in the context of Sezary syndrome. Our results demonstrate that RT39 exerts an efficient and specific cytotoxic activity towards Sezary cells, while sparring the other immune cell populations, both ex vivo on primary patient cells and in vivo in preclinical Sezary syndrome murine models. Mechanistic studies revealed that RT39 induces membranolysis of Sezary cells by means of binding to p21-activated kinase 1 (PAK1) in the context of Sezary cells plasma membrane, where PAK1 is overexpressed. Preliminary pharmacokinetic studies in mice indicated that, after intravenous, subcutaneous or intraperitoneal administration, the half-life of the peptide is of the order of 30 minutes, but the elimination phase is very prolonged with high AUCs, indicative of durable exposure. Along with excellent tolerability in mice, our preclinical results support the development of RT39 as a new therapeutic strategy in Sezary syndrome.

To the editor: Sezary syndrome (SS) is a rare, aggressive leukemic form of primary cutaneous T-cell lymphoma.[1][1] The diagnostic criteria associate a clonal T-cell receptor (TCR) rearrangement with peripheral Sezary cell (SC) counts of ≥1 G/L, an increased CD4/CD8 ratio of ≥10, CD4+CD7−

Purpose: KIR3DL2 is a recently discovered marker of the malignant clonal cell population in Sézary syndrome. We intended to evaluate the expression of KIR3DL2 on blood T cells as a diagnostic, prognostic, and follow-up marker of Sézary syndrome. Experimental Design: Sixty-four patients diagnosed with Sézary syndrome were included in this monocentric study. We collected the percentage of KIR3DL2+ cells among CD3+ T cells, obtained by flow cytometry, and other classical diagnostic criteria for Sézary syndrome at diagnosis and during the follow-up. Results: Compared with the classical diagnostic factors, KIR3DL2 was the most sensitive diagnostic factor for Sézary syndrome. Univariate and multivariate analyses established that an eosinophil cell count >700/mm3 and a percentage of KIR3DL2+ cells within the CD3+ T cells >85% at diagnosis were associated with a significantly reduced disease-specific survival. Moreover, KIR3DL2 immunostaining allowed the assessment of treatment efficiency and specificity toward tumor cells, the detection of the residual disease following treatment, and the occurrence of relapse, even though patients clinically experienced complete remission and/or undetectable circulating Sézary cells by cytomorphologic analysis. Conclusions: We show that KIR3DL2 expression is the most sensitive diagnostic criterion of Sézary syndrome when compared with all other available biological criteria. It also represents the best independent prognostic factor for Sézary syndrome–specific death and the most relevant feature for the follow-up of Sézary syndrome, showing the invasion of the functional lymphocytes pool by Sézary cells. KIR3DL2 therefore represents a valuable tool for routine use as a clinical parameter at diagnosis, for prognosis and during patient follow-up. Clin Cancer Res; 23(14); 3619–27. ©2017 AACR.

Background The early diagnosis of Sezary syndrome (SS) is challenging. Loss of CD7 and CD26 expression on CD4+ T cells is the currently used criterion in the initial diagnosis and staging of patients with SS. Objectives Our aim was to evaluate the respective value of CD26, CD7 and KIR3DL2 expression on CD4+ T cells and total lymphocytes at initial diagnosis of SS. Methods This prospective study included 254 patients with clinical features consistent with cutaneous T-cell lymphoma seen at our institution between March 2014 and February 2019. Peripheral blood analysis by flow cytometry was performed for each patient at the time of diagnosis and during follow-up. The diagnosis of SS was based on ISCL/EORTC criteria. Results The presence of KIR3DL2+ Sezary cells (SCs) ≥ 200 μL-1 correlated with the diagnosis of SS, with sensitivity of 88·6% and specificity of 96·3%. All 154 patients with either inflammatory skin disease or other haematological disease had KIR3DL2+ cells Conclusions KIR3DL2 expression on T cells is highly specific and helps the early diagnosis of SS, especially in those patients with lymphopenia. What's already known about this topic? In the ISCL/EORTC cutaneous T-cell lymphoma (CTCL) categorization of blood involvement (B0-B2), B2 is defined as a T-cell receptor clonal rearrangement in blood, associated with high blood-smear Sezary cell (SC) count. Flow cytometry was developed to circumvent interobserver variability of SC manual counts; however, it mostly relies on detection of cells lacking CD7 and/or CD26 expression. We previously reported the reliability of KIR3DL2 as the first positive SC marker. What does this study add? Based on our analysis of 254 patients, we propose that KIR3DL2 be added to the ISCL/EORTC criteria for initial diagnosis of Sezary syndrome (SS) and B2 staging. This marker improved sensitivity of SS B2-stage CTCL diagnosis with a specificity > 95%, especially for patients with lymphopenia. We found KIR3DL2 helped early diagnosis of SS and was more reliable than CD26 in assessing blood tumour burden during therapy. What is the translational message? SC quantification is the major means of staging at initial diagnosis and monitoring blood tumour burden in a clinical trials setting. We recommend using a threshold value of KIR3DL2+ SCs ≥ 200 μL-1 or KIR3DL2+ SCs/lymphocytes ≥ 10% in the diagnostic criteria of SS and propose a novel algorithm for CTCL B2 blood staging.

We previously identified the NK cell receptor KIR3DL2 as a valuable diagnostic and prognostic marker for the detection of the tumoral T cell burden of Sezary syndrome (SS) patients. However, the function of this receptor on the malignant T lymphocyte population remained unexplored. We here demonstrate that engagement of KIR3DL2 by its recently identified ligand CpG oligodeoxynucleotide (ODN) induces the internalization of the receptor and leads to a caspase-dependent apoptosis of malignant T cells. This process of cellular death is correlated to a dephosphorylation of the transcription factor STAT3 (signal transducer and activator of transcription 3), which is found constitutively phosphorylated and activated in Sezary cells. Our results indicate that KIR3DL2 can directly promote SS malignant cell death through the use of CpG ODN.

Advanced cutaneous T-cell lymphoma (CTCL) remains an unmet medical need, which lacks effective targeted therapies. In this study, we report the development of IPH4102, a humanized monoclonal antibody that targets the immune receptor KIR3DL2, which is widely expressed on CTCL cells but few normal immune cells. Potent antitumor properties of IPH4102 were documented in allogeneic human CTCL cells and a mouse model of KIR3DL2+ disease. IPH4102 antitumor activity was mediated by antibody-dependent cell cytotoxicity and phagocytosis. IPH4102 improved survival and reduced tumor growth in mice inoculated with KIR3DL2+ tumors. Ex vivo efficacy was further evaluated in primary Sézary patient cells, sorted natural killer–based autologous assays, and direct spiking into Sézary patient peripheral blood mononuclear cells. In these settings, IPH4102 selectively and efficiently killed primary Sézary cells, including at unfavorable effector-to-target ratios characteristic of unsorted PBMC. Together, our results offer preclinical proof of concept for the clinical development of IPH4102 to treat patients with advanced CTCL. Cancer Res; 74(21); 6060–70. ©2014 AACR.

CD160 is a GPI-anchored Ig-like receptor identified by the BY55 mAb on human circulating CD56dim+ NK cells and TCRγδ lymphocytes. In addition, while most intestinal T lymphocytes express it, only a minor circulating CD4+ or CD8+ T lymphocyte subset is CD160+. Here we describe a population of CD4+CD160+ human blood T lymphocytes of circulating cutaneous T cells. These rare T lymphocytes represent 2.1 ± 1.9% of the circulating CD3+CD4+ T cells, coexpress CD8αα, CD244, and perforin but lack CD28 expression, a phenotype corresponding to effector memory cytotoxic T-lymphocytes. Functional studies further confirmed their cytotoxic potential. These cells lack αEβ7 integrin and CCR7 expression but do express skin-addressing molecules CLA, and CCR4. In normal human skin, CD4+CD160+ cells represent 34.6 ± 14.7% of the CD4+ T lymphocytes extracted by collagenase treatment. These T cells coexpress CLA (81 ± 13.6%), CCR4 (62.3 ± 15.9%), and some CD8αα (19.6 ± 13%) or CCR7 (24.4 ± 11.7%) expression. Cutaneous T-cell lymphoma cells express the natural killer receptor KIR3DL2 (CD158k) used as a tumor marker. Not only we confirmed the expression of this marker in the blood and/or skin of mycosis fungoides patients but we also show for the first time CD158k expression (often associated with CD160) on cutaneous CD4+ T cells from healthy individuals (25.3 ± 15%). Therefore, CD4+CD160+ T cells expressing CD158k might represent specialized cutaneous lymphocytes devoted to immune surveillance, from which could originate cutaneous T-cell lymphomas such as mycosis fungoides. © 2014 International Society for Advancement of Cytometry

Introduction KIR3DL2 est un recepteur NK exprime par 85 % des lymphomes T cutanes (LTC). IPH4102 est un anticorps (Ac) monoclonal « first-in-class » anti-KIR3DL2 qui deplete les cellules par des mecanismes d’ADCC et d’ADCP (cytotoxicite et phagocytose dependantes de l’anticorps). Il a ete teste pour la premiere fois chez l’homme dans une etude de phase I afin d’evaluer sa tolerance et de determiner la dose maximale toleree (DMT) et la dose recommandee pour une etude de phase 2 (RP2D). Les objectifs secondaires comportaient des etudes pharmacocinetiques et l’evaluation de l’efficacite clinique et biologique. Patients et methodes Les malades inclus etaient des LTC de stade avance refractaires a au moins 2 lignes de traitement systemique, dont les cellules tumorales exprimaient KIR3DL2 dans la peau et/ou le sang. L’escalade de dose comportait dix niveaux (0,0001 a 10 mg/kg). L’Ac etait administre par voie IV toutes les semaines pendant 4 semaines, toutes les 2 semaines jusqu’a la semaine 26 puis toutes les 4 semaines. Les malades pouvaient recevoir des doses croissantes et etaient traites jusqu’a progression ou survenue d’une toxicite. L’efficacite clinique etait evaluee selon les criteres internationaux. Les lymphocytes KIR3DL2 etaient monitores dans la peau par immunohistochimie et dans le sang par cytometrie de flux. La maladie residuelle etait evaluee par « deep sequencing » du recepteur T pour l’antigene. Resultats Vingt-cinq patients ont ete inclus : 20 syndromes de Sezary (SS), 4 mycosis fongoides (MF) et un LTC CD4+ NOS. L’âge median etait de 71 ans (42–90) et le nombre median de traitements precedents de 4 (2–14). Dix-neuf malades ont recu des doses croissantes d’Ac. Aucune toxicite limitante n’a ete observee et la DMT n’a pas ete atteinte. Six patients ont presente des evenements indesirables (EI) de grade 3, dont 2 lies au traitement et 2 des EI de grade 4 non lies au traitement. Les EI etaient pour la majorite des evenements de bas grade frequents lors de perfusions d’Ac. Le taux de reponses globales etait de 50 % pour les SS, dont 65 % de reponses dans le sang incluant 25 % de reponses completes. Les scores de prurit etaient tres ameliores chez tous les malades repondeurs et stables. Les lymphocytes KIR3DL2 ont ete progressivement et durablement depletes dans la peau et le sang. L’evaluation de la maladie residuelle a montre la disparition totale et durable du clone tumoral dans le sang et la peau. Une posologie de 10 mg/kg a ete choisie pour la phase d’extension. Conclusion Ces resultats montrent une excellente tolerance et une efficacite clinique tres prometteuse d’IPH4102 chez des malades ayant un LTC refractaire a plusieurs traitements systemiques. Une phase d’expansion de 15 malades ayant un syndrome de Sezary est en cours, les malades recevant une « flat dose » de 750 mg.

TIGIT is a T cell co-inhibitory receptor recently described as a key checkpoint driving tumor cell immunosuppression. It is predominantly expressed on CD4+ Tregs, CD8+ T cells and NK cells from healthy individuals but further upregulated in cancer patients where it frequently co-expresses with exhaustion markers such as PD-1. TIGIT cognate receptors are members of the poliovirus receptors, among which CD155 has the highest affinity for TIGIT. They are expressed on antigen presenting cells but also on tumor cells which provides a strong rationale for blocking TIGIT as a therapeutic approach to reverse T or NK cell dysfunction linked with cancer progression. To substantiate the importance of TIGIT as an immunotherapy target, we used flow cytometry and immunohistochemistry (IHC) to extensively characterize TIGIT and CD155 expression in both healthy donors and cancer patients. We initially confirmed TIGIT expression on multiple immune subsets from healthy donors. Similar flow cytometry analysis performed on matched circulating and tumor-infiltrating immune populations from 15 cancer patients highlighted the global overexpression of TIGIT associated with cancer. Interestingly, ex vivo polyfunctional analysis of cytokine release strongly supported the immunosuppressed character of infiltrated TIGIT positive CD4+ and CD8+ T cells. Finally, combining receptor density with frequency of positive cells led to the interesting observation that tumor-infiltrating Tregs represents the population with the highest TIGIT expression, confirming the opportunity to preferentially target that suppressive population within the tumor microenvironment. These findings on Tregs were further confirmed by IHC. TIGIT was expressed in 10 out of the 11 lung adenocarcinoma tissues analysed. Both Tregs and CD8+ T cells express TIGIT, with Tregs showing a higher proportion of TIGIT+ cells. Finally, direct expression of TIGIT on tumor cells was also found on several haematological malignancies, opening the door for anti-TIGIT therapies to act directly on tumor cells besides revigoration of the immune system. The presence of TIGIT ligands within the tumor microenvironment was also assessed and confirmed using both flow cytometry and IHC. CD155 expression was analysed by IHC in both normal individuals (n=90) and cancer patient tissues from 9 different cancer indications (n=284). In cancer tissues, CD155 is mostly expressed by tumor cells, ranging from a median percentage of 50 % for pancreatic cancer to 2 % for cervix cancer. Cancer samples with the highest CD155 expression are pancreatic, prostate, kidney, gastric and colon cancers. CD155 expression is always lower in normal tissues compared to their cancer counterparts except for lung. Together, these data strongly support the relevance of targeting TIGIT in immuno-oncology and pave the way to select the ideal indications where patients could benefit from such a therapy. Citation Format: Noémie Wald, Marjorie Mercier, Julia Cuende, Florence Nyawouame, Shruthi Prasad, Margreet Brouwer, Erica Houthuys, Anne Marie-Cardine, Martine Bagot, Grégory Driessens, Véronique Bodo, Catherine Hoofd. TIGIT pathway phenotyping sheds light on promising strategies to restore anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4969.

Differentiation between Sezary syndrome and erythrodermic inflammatory dermatoses can be challenging, and a number of studies have attempted to identify characteristic immunophenotypic changes and molecular biomarkers in Sezary cells that could be useful as additional diagnostic criteria. In this European multicenter study, the sensitivity and specificity of these immunophenotypic and recently proposed but unconfirmed molecular biomarkers in Sezary syndrome were investigated. Peripheral blood CD4 + T cells from 59 patients with Sezary syndrome and 19 patients with erythrodermic inflammatory dermatoses were analyzed for cell surface proteins by flow cytometry and for copy number alterations and differential gene expression using custom-made quantitative PCR plates. Experiments were performed in duplicate in two independent centers using standard operating procedures with almost identical results. Sezary cells showed MYC gain (40%) and MNT loss (66%); up-regulation of DNM3 (75%), TWIST1 (69%), EPHA4 (66%), and PLS3 (66%); and down-regulation of STAT4 (91%). Loss of CD26 (≥80% CD4 + T cells) and/or CD7 (≥40% CD4 + T cells) and combination of altered expression of STAT4 , TWIST1 , and DNM3 or PLS3 could distinguish, respectively, 83% and 98% of patients with Sezary syndrome from patients with erythrodermic inflammatory dermatoses with 100% specificity. These additional diagnostic panels will be useful adjuncts in the differential diagnosis of Sezary syndrome versus erythrodermic inflammatory dermatoses.

Resume Le syndrome de Sezary (SS) represente 3 % des lymphomes T cutanes et appartient aux lymphomes T cutanes agressifs, avec un taux de survie a cinq ans de 24 %. Selon l’European organization for research and treatment of cancer (EORTC), le SS est defini par la presence d’une erythrodermie, d’adenopathies diffuses, de lymphocytes T atypiques sanguins (> 1000/mm3) et d’un clone T dominant dans le sang, la peau et les ganglions. Un marqueur specifique des lymphocytes T atypiques tumoraux appeles cellules de Sezary a ete identifie en 2001, le recepteur KIR3DL2 (CD158k), qui permet un diagnostic plus specifique de SS, et dont le taux est bien correle a l’evolution clinique de la maladie. Sur le plan therapeutique, de nouvelles molecules font l’objet d’essais therapeutiques et representent des perspectives d’amelioration du pronostic de la maladie. Nous proposons une mise au point sur cette pathologie qui fait actuellement l’objet de travaux scientifiques tant physiopathologiques que therapeutiques, avec de nouveaux espoirs de therapies ciblees.

IL-1 receptor accessory protein (IL-1RAcP) is the second subunit required to form a functional receptor complex for IL-1α and β, IL-1F6, IL-1F8, IL1-F9 and IL-33. While it does not directly interact with the cytokines, IL-1RAcP is necessary to mediate signal transduction. We previously reported a monoclonal antibody with an unknown specificity, termed AY19, that was capable to induce a significant increase in the size of CFU-GM colonies when added to cultures of human cord blood CD34(+) hematopoietic progenitors. Here we demonstrate that AY19 mAb recognizes IL1-RAcP. We show that this adaptor molecule is significantly present on peripheral blood monocytes and lymphocytes including CD4(+) and CD8(+) T lymphocytes, B and NK cells. Interestingly, its expression is found increased on CD127(low)CD4(+)CD25(high) T cells when compared to CD127(low)CD4(+)CD25(-) T cell subset, suggesting that the level of IL-1RAcP membrane expression could allow to distinguish within CD127(low)CD4(+) T lymphocytes the CD25(high) T regulatory subset from conventional CD25(-) T lymphocytes. Functional studies reveal that addition of AY19 mAb enhances the proliferation of peripheral blood mononuclear cells (PBMC) obtained with mitogenic concentrations of PMA. Interestingly, we found that although AY19 mAb does not increase the optimal PBMC proliferation induced by a mitogenic pair of anti-CD2 mAbs it prolongs their time of proliferation. Thus, these results indicate that the anti-IL-1RAcP mAb AY19 exhibits unique functional properties by triggering co-stimulatory signals in lymphocytes.

The natural cytotoxicity receptors NKp30, NKp44, and NKp46 were identified as activating receptors mainly expressed by natural killer (NK) lymphocytes. In this study we show that peripheral blood malignant CD4(+) T lymphocytes from patients with Sézary syndrome, an aggressive form of cutaneous T-cell lymphoma, express NKp46 at their cell surface. Although NKp46 does not behave as an independent functional receptor, its engagement provides a strong inhibiting signal on the malignant T lymphocyte CD3-induced proliferation. We show that this inhibition is correlated with a decreased phosphorylation of the CD3ζ chain associated with NKp46 and/or the TCR/CD3 complexes. Our results reveal that in addition to KIR3DL2/CD158k expression, NKp46 could represent an additional marker on the circulating malignant T lymphocytes of Sézary patients, where it displays an as yet unreported function of inhibitory co-receptor able to interfere with the processes governing their CD3-dependent proliferation.

Introduction Cutaneous T-cell lymphoma (CTCL), a heterogeneous group of rare non-Hodgkin's lymphomas, is characterized by infiltration of malignant clonally expanded T lymphocytes in the skin and is very challenging to treat at advanced stages. Around 65% of the CTCL cases are mycosis fungoides (MF), which usually starts as indolent disease with accumulation of neoplastic resident memory T lymphocytes in the skin, evolving from patch to plaque and later to tumor stage with poor prognosis. Sézary syndrome (SS) involves erythroderma in the skin, malignant circulating clone, and lymphadenopathy. The CTCL pathogenesis is incompletely understood but suggests a complex interplay between neoplastic T cells and the tumor microenvironment. Glycoengineered ARGX-110 potently blocks CD70/CD27 signaling, which is thought to inhibit evasion of tumor immune surveillance as well as tumor cell proliferation and survival. The ARGX-110 effector functions, i.e., complement-dependent cytotoxicity (CDC), antibody-dependent cellular phagocytosis (ADCP), and enhanced antibody-dependent cellular cytotoxicity (ADCC), efficiently kill CD70-expressing tumor cells. With only limited expression in normal tissues and strong expression on tumor cells, CD70 is a very attractive target for antibody-based therapy in cancer. The role of CD70 and treatment with ARGX-110 was further investigated as part of a Phase 1/2 study in advanced CTCL patients. Methods As part of a non-randomized, open-label, multicenter Phase 1/2 study (NCT01813539), a total of 27 patients with CD70-expressing advanced CTCL of different subtypes and stages were included for determination of the optimal dose, to evaluate exploratory efficacy and safety, and to characterize pharmacokinetics and biomarkers of drug activity (immunohistochemistry [IHC] staining for drug- and disease-related markers, profiling of malignant and non-malignant immune cells by flow cytometry, quantitative polymerase chain reaction [qPCR] and Nanostring). ARGX-110 was administered intravenously at 1 (N=11) or 5 mg/kg (N=16) every three weeks (Q3W). Adverse events were graded according to NCI-CTCAE v. 4.03. Results ARGX-110 was safe and well tolerated at both 1 and 5 mg/kg in heavily treated CTCL patients with a median age of 67 years (range: 25-84 years). At cut-off (26th June 2018), a total of 110 treatment-emergent adverse events (TEAE) were reported in 27 patients of which 12 were grade 3-5; 40 events in 18 patients were considered drug-related by the investigator. The most common TEAE was pyrexia (7 events in 5 patients), and infusion-related reactions (IRRs) was the most common drug-related TEAE. The best response was 1 complete response (CR), 5 partial response (PR), and 7 stable disease (SD) in 26/27 patients evaluable for response, meaning overall response (ORR) in 23% of the patients and disease stabilization in 27% of the patients. The mean duration on the study was around 4.8 months. Two patients were still on the study at cut-off: 1 patient (subcutaneous panniculitis-like T-cell lymphoma, 24 months) with CR and 1 SS patient with PR (6.5 months). Pharmacokinetics (PK) revealed a mean half-life of 8-12 days, in line with previously published data. The 5 mg/kg dose was chosen as the recommended phase 2 dose (RP2D) based on PK and immunogenicity data. IHC staining for drug- and disease-specific markers showed results in line with clinical reduction of modified Severity Weighted Assessment Tool (mSWAT) and regression from plaque to patch stages. Molecular and cellular profiling indicated a complex interplay between the tumor and the microenvironment, in which CD70/CD27 plays a role. Conclusions ARGX-110 was safe and well tolerated at both dose levels (1 and 5 mg/kg) in advanced CD70-expressing CTCL patients. Clinical anti-tumor activity in patients with various types of CTCL was observed after treatment with ARGX-110, indicating ARGX-110 as a safe and promising treatment option for advanced CTCL. Disclosures Bagot: Kyowa Kirin: Membership on an entity's Board of Directors or advisory committees; Actelion: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Innate Pharma: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Maerevoet:Karyopharm: Membership on an entity's Board of Directors or advisory committees; takeda: Membership on an entity's Board of Directors or advisory committees, Other: travel grant; roche: Other: travel grant; abbvie: Membership on an entity's Board of Directors or advisory committees, Other: travel grant. Zinzani:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bayer: Membership on an entity's Board of Directors or advisory committees; PFIZER: Honoraria, Membership on an entity's Board of Directors or advisory committees; PFIZER: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; MSD: Honoraria, Speakers Bureau; Janssen: Honoraria, Speakers Bureau; Celltrion: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Astra Zeneca: Speakers Bureau; TG Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees; Merck: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Merck: Honoraria, Membership on an entity's Board of Directors or advisory committees; Verastem: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; TG Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; SERVIER: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Morschhauser:Janssen: Other: Scientific Lectures; Epizyme: Consultancy; Roche: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees. Ribrag:Incyte Corporation: Consultancy; Amgen: Research Funding; Infinity: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; epizyme: Consultancy, Honoraria; NanoString Technologies: Consultancy, Honoraria; Roche: Honoraria, Other: travel; Servier: Consultancy, Honoraria; pharmamar: Other: travel; argenX: Research Funding; BMS: Consultancy, Honoraria, Other: travel; MSD: Honoraria. Dalle:Kyowa Hakko Kirin Pharmaceutical: Research Funding. Cayuela:Cepheid: Other: financial sponsor to attend John Goldman Conference 2017. Hultberg:argenx: Employment. Gandini:argenx: Employment. Moshir:argenx: Employment. Delahaye:argenx: Employment. Zabrocki:argenx: Employment. Silence:argenx: Consultancy. Van Rompaey:argenx: Employment. De Haard:argenx: Employment. Leupin:argenx: Employment.

Background CD158k/KIR3DL2 is a specific marker for Sezary cells which can be used to diagnose Sezary syndrome (SS) in erythrodermic patients with abnormal circulating T cells. Objectives To evaluate the suitability of CD158k/KIR3DL2 for detecting and evaluating blood tumour load during the follow up of patients with SS. Methods The absolute CD3+ CD158k+ lymphocyte count was compared with the absolute count of cytomorphological Sezary cells and was correlated with clinical flares in a cohort of patients with SS. Twenty-five patients were included in the study and 48 blood samples were analysed. Results The absolute count of CD3+ CD158k+ cells strongly correlated with the absolute count of atypical circulating cells (r = 0.97, P < 10(-15)). The CD3+ CD158k+ lymphocyte cell count was in eight cases more sensitive than cytomorphology for detecting atypical circulating cells especially for small-sized tumour cells. The tumour burden evaluated by CD3+ CD158k+ immunostaining was significantly associated with clinical flare (P < 10(-4)). Conclusions CD3+ CD158k+ phenotyping is a reliable and objective test to monitor the blood tumour burden in patients with SS under systemic therapy.

CD160 is a GPI-anchored lymphocyte surface receptor in which expression is mostly restricted to the highly cytotoxic CD56dimCD16+ peripheral blood NK subset. We previously reported that MHC class I (MHC-I) molecules bind to CD160 receptors on circulating NK lymphocytes and that this interaction triggers their cytotoxic activity and cytokine production. We also observed that CD160 surface expression on NK cells is down-modulated upon activation with PMA or IL-2. In this study, we further report that short-time incubation of NK lymphocytes with IL-15 converts the membrane-bound CD160 to a soluble form through a proteolytic cleavage involving a metalloprotease. Thus, CD160 is no longer detected at the cell surface, but can be immunoprecipitated from the NK cell culture medium. Interestingly, CD160 transcript remains highly expressed during the process of protein shedding. In addition, we demonstrate that CD160 mRNA synthesis can be induced in CD56bright separated lymphocytes following exposure to IL-15. By producing a Flag-tagged soluble CD160 protein, we establish that its binding to MHC-I molecules results in the inhibition of the cytotoxic CD8+ T lymphocyte activity and of the CD160-mediated NK cell cytotoxicity. Thus, we show that activated NK lymphocytes release a soluble form of CD160 that functionally impairs the MHC-I-specific cytotoxic CD8+ T lymphocyte responsiveness.

Cutaneous T-cell lymphomas (CTCL) represent a heterogenous group of diseases primarily involving the skin. Among them, Sezary syndrome (SS) and transformed mycosis fungoides (MF) are aggressive forms of CTCL that currently lack effective therapy. The search for new monitoring and therapeutic targets in this context is in dire need. KIR3DL2 (CD158k) is potentially one of these targets expressed by the tumor cell population when only expressed by few normal T and NK lymphocytes. Recently IPH4102 a humanized monoclonal antibody was developed to selectively bind and deplete KIR3DL2-expressing tumor cells. It acts by recruiting autologous NK cells and macrophages via Fc receptors to target elimination of tumor cells. Moreover, engagement of KIR3DL2 by its recently identified ligand CpG oligodeoxynucleotides induces its internalization and the triggering of a caspase dependent apoptosis of the malignant T cells. These two therapeutic strategies targeting KIR3DL2 are actively pursued toward the development of clinical studies for CTCL.

CD245 is a human surface antigen expressed on peripheral blood lymphocytes, initially delineated by two monoclonal antibodies DY12 and DY35. Until now, CD245 molecular and functional characteristics remained largely unknown. We combined immunological and proteomic approaches and identified CD245 as the unconventional myosin 18A, a highly conserved motor enzyme reported as a receptor for the surfactant protein A (SP-A), that plays a critical role in cytoskeleton organization and Golgi budding. We report that the recruitment of CD245 strongly enhanced NK cell cytotoxicity. Further, we show that the enhancement of the NK lymphocytes killing ability toward CD137-ligand expressing target cells could result from the induction of CD137 expression following CD245 engagement. The SP-A receptor could therefore represent a novel and promising target in cancer immunotherapy.

Identification of malignant Sézary cells by T-cell receptor (TCR) clonality studies is routinely used for the diagnosis of Sézary syndrome, but T-cell clones expressed in a single patient have never been accurately characterized. We previously reported that CD158k expression delineates Sézary syndrome malignant cells, and, more recently, we identified vimentin at the surface membranes of Sézary cells and normal activated lymphocytes. In the present study, T-cell clones from 13 patients with Sézary syndrome were identified by immunoscopy and further characterized in the blood according to their TCR Vβ, CD158k, and vimentin cell-surface expression. We found in most patients a unique malignant T-cell clone that coexpressed CD158k and vimentin and that, when patients were tested, was also present in the skin. However, in some patients we detected the presence of a nonmalignant circulating clone expressing high amounts of vimentin and lacking CD158k. These results indicate that clonal expansion may originate from circulating malignant and nonmalignant CD4+ T cell populations in patients with Sézary syndrome. Identification of the malignant cells in Sézary syndrome cannot be achieved by T-cell clonality studies or by TCR Vβ monoclonal antibody (mAb) analysis alone; it also relies on CD158k phenotyping.

Circulating human natural killer (NK) lymphocytes have been functionally defined by their ability to exert cytotoxic activity against MHC class I-negative target cell lines, including K562. Therefore, it was proposed that NK cells recognized the “missing self.” We show here that the Ig-like CD160 receptor expressed by circulating CD56dim+NK cells or IL-2-deprived NK cell lines is mainly involved in their cytotoxic activity against K562 target cells. Further, we report that HLA-C molecules that are constitutively expressed by K562 trigger NK cell lysis through CD160 receptor engagement. In addition, we demonstrate, with recombinant soluble HLA-Cw3 and CD160 proteins, direct interaction of these molecules. We also find that CD158b inhibitory receptors partially interfere with CD160-mediated cytotoxicity, whereas CD94/CD159a and CD85j have no effect on engagement with their respective ligands. Thus, CD160/HLA-C interaction constitutes a unique pathway to trigger NK cell cytotoxic activity.

Introduction Nous avons precedemment montre la fiabilite du marqueur CD158k/KIR3DL2 pour l’identification et la caracterisation des cellules de Sezary (CS), et avons ainsi mis en evidence une heterogeneite inattendue de ces cellules. Materiel et methodes Quarante-sept echantillons sanguins de patients atteints de syndrome de Sezary (SS), ainsi que 16 biopsies cutanees prelevees le meme jour ont ete analyses en cytometrie en flux pour l’expression de marqueurs de maturation (CD45RA, CCR7, CD27, CD95) et de lymphocytes T residents memoires (TRM : CD69, CD103). La concordance entre les lymphocytes T CD158k+ et le clone Vβ connu du TCR a ete verifiee lorsque l’anticorps etait disponible. Le transcriptome de 3 patients a ete realise sur CS sanguines triees de divers phenotypes, en parallele de populations T CD4+ de 3 donneurs sains (DS), sur puces Affymetrix HTA 2.0. Resultats Quarante-trois pour cent des echantillons sanguins presentaient une heterogeneite des types naifs/memoires de CS, avec 15 % de phenotypes TSCM (stem cell memory), precedemment decrits chez le sujet sain comme doues de fortes capacites d’auto-renouvellement et de differenciation, et impliques comme reservoir tumoral dans le lymphome T de l’adulte (ATL). Dans la peau, les CS presentaient des profils de maturation plus avances, exclusivement memoires, avec des marqueurs de TRM. Afin de relier ces phenotypes a un reel profil de T naif/memoire, une etude transcriptomique a ete realisee apres tri de differents types de CS circulants naif/TSCM/memoire, en parallele des sous-populations correspondantes chez des DS. Les resultats de 3 patients, apres elimination des genes impliques dans les voies d’activation oncogenique, ont mis en evidence une signature transcriptomique naive/memoire proche des sous-types T CD4+ connus : un patient presentait des CS comparables aux TSCM de DS ; un deuxieme proche de centrales memoires (TCM) ; un troisieme presentait une heterogeneite intra-clonale, avec des CS CD45RA+CD95− proches de TSCM, et CD45RA−CD95− proches de TCM. En revanche, ces CS differaient de leurs homologues naifs/memoires des DS par des expressions anormales de transcrits impliques dans la reponse aux cytokines et dans la differenciation lymphocytaire T. Discussion Nos resultats confirment l’heterogeneite intra- et inter-individuelle des CS en termes de phenotypes et de profils de maturation. Chez un meme patient les phenotypes des CS cutanees presentaient des caracteristiques plus matures que les CS circulantes. Ces phenotypes ont pu etre relies dans le sang chez 3 patients, aux profils transcriptomiques naifs/memoires etablis chez les sujets sains, notamment TSCM, remettant en cause le dogme selon lequel les CS seraient exclusivement de type TCM. Conclusion Ces donnees contribuent a une meilleure comprehension de l’origine et de l’evolution des CS au cours du SS, ainsi que des interactions entre les compartiments sanguins et cutanes, et pourraient permettre de definir des cibles cellulaires pertinentes pour les immunotherapies du futur.

To the editor: Sezary syndrome (SS) is an aggressive leukemic and erythrodermic variant of cutaneous T-cell lymphoma characterized by the presence of a clonal T-lymphocyte population in the skin, lymph nodes, and peripheral blood. We previously reported that KIR3DL2 represents a specific cell