Transfection of FcγRIIIa (CD16) Alone Can Be Sufficient To Enable Human αβTCR T Lymphocytes To Mediate Antibody-Dependent Cellular Cytotoxicity

International audienceTo combine the immune potential of T cells and Ab therapy, we and others have previously shown that T cells transduction with a fusion receptor that binds the Fc portion of human Ig enable them to mediate Ab-dependent cellular cytotoxicity (ADCC). The fusion receptors previously described included the FcgRIIIa (CD16) receptor coupled to different chains intended to translate the signal. In this work, we questioned whether the transfection of CD16 alone into T human lymphocytes and NK cells could be sufficient for CD16 expression and function, or whether the cotransfection of a transducing chain was mandatory. Our results demonstrated that: 1) transfection of CD16 alone into a human NK cell line and primary T cells can be sufficient for CD16 expression and function; 2) cotransfection of CD3z or FceRIg increased CD16 expression; 3) yet this increased CD16 expression increased the ADCC score only for trastuzumab, not for rituximab or cetuximab; and 4) compared with that of peripheral NK cells, ADCC scores by autologous CD16-transfected T cells ranked differently according to the opsonized target cell. Together, these results showed that neither the use of a fusion receptor nor the cotransfection of a transducing chain is mandatory to transfer the ADCC function to human lymphocytes. Thus, depending on the effector/Ab/target combination considered, transfection of CD16 alone can be sufficient to enable T cells to mediate ADCC. In the context of immunotherapy, such a strategy is by nature safer than the use of a chimeric receptor, and is freely available

In Vitro and In Vivo Comparison of Lymphocytes Transduced with a Human CD16 or with a Chimeric Antigen Receptor Reveals Potential Off-Target Interactions due to the IgG2 CH2-CH3 CAR-Spacer

International audienceThe present work was designed to compare two mechanisms of cellular recognition based on Ab specificity: firstly, when the anti-HER2 mAb trastuzumab bridges target cells and cytotoxic lymphocytes armed with a Fc receptor (ADCC) and, secondly, when HER2 positive target cells are directly recognized by cytotoxic lymphocytes armed with a chimeric antigen receptor (CAR). To compare these two mechanisms, we used the same cellular effector (NK-92) and the same signaling domain (Fcí µí¼RIí µí»¾). The NK-92 cytotoxic cell line was transfected with either a Fcí µí»¾RIIIa-Fcí µí¼RIí µí»¾ (NK-92 CD16) or a trastuzumab-based scFv-Fcí µí¼RIí µí»¾ chimeric receptor (NK-92 CAR). In vitro, the cytotoxic activity against HER2 positive target cells after indirect recognition by NK-92 CD16 was always inferior to that observed after direct recognition by NK-92 CAR. In contrast, and somehow unexpectedly, in vivo, adoptive transfer of NK-92 CD16 + trastuzumab but not of NK-92 CAR induced tumor regression. Analysis of the in vivo xenogeneic system suggested that the human CH2-CH3 IgG2 used as a spacer in our construct was able to interact with the FcR present at the cell surface of the few NSG-FcR+ remaining immune cells. This interaction, leading to blockage of the NK-92 CAR in the periphery of the engrafted tumor cells, stresses the critical role of the composition of the spacer domain

Merkel cell carcinoma and cellular cytotoxicity: sensitivity to cellular lysis and screening for potential target antigens suitable for antibodydependent cellular cytotoxicity

The online version of this article ( https://doi.org/10.1007/s00262-018-2176-2) contains supplementary material, which is available to authorized users.International audienceThe recent success of checkpoint inhibitors in the treatment of Merkel cell carcinoma (MCC) confirms that MCC tumors can be immunogenic. However, no treatment directly targeting the tumor is available for use in combination with these checkpointinhibitors to enhance their efficacity. This study was carried out to characterize MCC line sensitivity to cellular lysis and to identify cell surface antigens that could be used for direct targeting of this tumor. For five representative MCC lines, the absence or low expression of MICA, MICB, HLA-I, and ICAM-1 was associated with low level of recognition by NK cells and T lymphocytes. However, expression of HLA-I and ICAM-1 and sensitivity to cellular lysis could be restored or increased after exposure to INFγ. We tested 41 antibodies specific for 41 different antigens using a novel antibody-dependent cellular cytotoxicity (ADCC) screening system for target antigens. Anti-CD326 (EpCAM) was the only antibody capable of inducing ADCC on the five MCC lines tested. Because MCC tumors are often directly accessible, local pharmacologic manipulation to restore HLA class-I and ICAM-1 cell surface expression (and thus sensitivity to cell lysis) can potentially benefit immune therapeutic intervention. In line with this, our observation that ADCC against EpCAM can induce lysis of MCC lines and suggests that therapeutic targeting of this antigen deserves to be explored further

The doubling potential of T lymphocytes allows clinical-grade production of a bank of genetically modified monoclonal T-cell populations

International audienceBackground aims. To produce an anti-leukemic effect after hematopoietic stem cell transplantation we have long considered the theoretical possibility of using banks of HLA-DP specific T-cell clones transduced with a suicide gene. For that application as for any others, a clonal strategy is constrained by the population doubling (PD) potential of T cells, which has been rarely explored or exploited. Methods. We used clinical-grade conditions and two donors who were homozygous and identical for all HLA-alleles except HLA-DP. After mixed lymphocyte culture and transduction, we obtained 14 HLA-DP–specific T-cell clones transduced with the HSV-TK suicide gene. Clones were then selected on the basis of their specificity and functional characteristics and evaluated for their doubling potential. Results. After these steps of selection the clone NAT-DP4[(TK)], specific for HLA-DPB1*04:01/04:02, which produced high levels of interferon-γ (IFNγ), tumor necrosis factor (TNF), interleukin-2 (IL-2) and granulocyte-macrophage colony-stimulating factor (GM-CSF), was fully sequenced. It has two copies of the HSV-TK suicide transgene whose localizations were determined. Four billion NAT-DP4[(TK)] cells were frozen after 50 PDs. Thawed NAT-DP4[(TK)] cells retain the potential to undergo 50 additional PDs, a potential very far beyond that required to produce a biological effect. This PD potential was confirmed on 6/16 additional different T-cell clones. This type of well-defined clone can also support a second genetic modification with CAR constructs. Conclusion. The possibility of choosing rare donors and exploiting the natural proliferative potential of T lymphocytes may dramatically reduce the clinical and immunologic complexity of adoptive transfer protocols that rely on the use of third-party T-cell populations

T cell repertoire and Epstein-Barr virus-specific T cell response in chronic active Epstein-Barr virus infection: a case study.

International audienceIn a patient with chronic active Epstein-Barr virus infection associated with vasculitis and fulminant CD4+ T cell lymphoproliferative disorder, we probed the peripheral blood mononuclear cells (PBMC) for the presence of an EBV-specific T cell repertoire and tested the possible relationship between the lymphocytic infiltrate and the EBV-specific T cell response. Our results give credence to the presence of an apparently normal EBV-specific memory T cell response after in vitro reactivation of the patient's PBMC with autologous infected B lymphoblastoid cell lines. In keeping with the characterization of the vasculitis, certain T cell subsets were detected after expansion of skin lesion-infiltrating lymphocytes and were found to be infected with EBV. These particular T cell expansions were neither the effectors nor the targets of the in vitro reactivated EBV-specific T cells, thus excluding a simple relationship between EBV, the skin lesions, and the T cell expansions frequently observed in these patients

Systems-level conservation of the proximal TCR signaling network of mice and humans

International audienceWe exploited traceable gene tagging in primary human T cells to establish the composition and dynamics of seven canonical TCR-induced protein signaling complexes (signalosomes) using affinity purification coupled with mass spectrometry (AP-MS). It unveiled how the LAT adaptor assembles higher-order molecular condensates and revealed that the proximal TCR-signaling network has a high degree of qualitative and quantitative conservation between human CD4 + and CD8 + T cells. Such systemslevel conservation also extended across human and mouse T cells and unexpectedly encompassed protein-protein interaction stoichiometry. Independently of evolutionary considerations, our study suggests that a drug targeting the proximal TCR signaling network should behave similarly when applied to human and mouse T cells. However, considering that signaling differences likely exist between the distal TCR-signaling pathway of human and mouse, our fast-track AP-MS approach should be favored to determine the mechanism of action of drugs targeting human T cell activation. An opportunity is illustrated here using an inhibitor of the LCK protein tyrosine kinase as a proof-of-concept

T-cell therapy using a bank of EBV-specific cytotoxic T cells: lessons from a phase I/II feasibility and safety study.

International audienceWe report herein the results we obtained and the limitations we experienced during the production and use of a bank of Epstein-Barr virus (EBV)-transformed human cytotoxic T lymphocytes (EBV-CTLs). To assess the feasibility and toxicity of this strategy, we selected and stored, in liquid nitrogen, 4 billion EBV-CTLs from each of the 13 selected donors. Subsequently, in a multicenter phase I/II study, 11 patients with EBV-associated lymphoma resistant to conventional treatments received 1-3 doses of 5 million EBV-CTLs/kg with 1-3 and 0-4 compatibilities for human leukocyte antigen (HLA)-I and HLA-II, respectively. Except for one event of fever after injection, no immediate or delayed toxicity, no graft versus host disease, and no graft rejection attributable to CTL infusion were observed. Three patients presented complete remission and 1 partial remission after treatment. Considering the clinical options currently available, and the constrains associated with CTL preparation and implementation, we conclude that CTL banks should consist of a reasonably small number of cell lines with documented specificities. This objective could be more easily achieved if the few homozygous donors for the most frequent HLA alleles of the targeted population could be made available for such a project