Advanced Flow Cytometry Assays for Immune Monitoring of CAR-T Cell Applications

Abstract

Adoptive immunotherapy using chimeric antigen receptor (CAR)-T cells has achieved successful remissions in refractory B-cell leukemia and B-cell lymphomas. In order to estimate both success and severe side effects of CAR-T cell therapies, longitudinal monitoring of the patient’s immune system including CAR-T cells is desirable to accompany clinical staging. To conduct research on the fate and immunological impact of infused CAR-T cells, we established standardized 13-colour/15-parameter flow cytometry assays that are suitable to characterize immune cell subpopulations in the peripheral blood during CAR-T cell treatment. The respective staining technology is based on pre-formulated dry antibody panels in a uniform format. Additionally, further antibodies of choice can be added to address specific clinical or research questions. We designed panels for the anti-CD19 CAR-T therapy and, as a proof of concept, we assessed a healthy individual and three B-cell lymphoma patients treated with anti-CD19 CAR-T cells. We analyzed the presence of anti-CD19 CAR-T cells as well as residual CD19+ B cells, the activation status of the T-cell compartment, the expression of co-stimulatory signaling molecules and cytotoxic agents such as perforin and granzyme B. In summary, this work introduces standardized and modular flow cytometry assays for CAR-T cell clinical research, which could also be adapted in the future as quality controls during the CAR-T cell manufacturing process.

Clinical and Product Features Associated with Outcome of DLBCL Patients to CD19-Targeted CAR T-Cell Therapy

“…From a technical perspective, different methods have been used to monitor PB CAR T-cells in patients, based either on PCR [ 40 , 41 , 42 ] or flow-cytometry [ 43 , 44 ]. As compared to PCR-based approaches, flow-cytometry CD4 and CD8 CAR + subpopulations can be distinguished [ 44 ].…”

Monitoring of Circulating CAR T Cells: Validation of a Flow Cytometric Assay, Cellular Kinetics, and Phenotype Analysis Following Tisagenlecleucel

“…A wide variety of CAR T cell detection methods have been described so far, which were mostly intended for basic science and clinical trial applications. Real-time PCR and flow cytometry have shown the most value for longitudinal monitoring of CAR T cells among them, as they are well established and widely available methods (23)(24)(25). Nevertheless, there are still few publications focusing on CAR-T cell monitoring with flow cytometry outside of clinical trials.…”

Recent Developments in Application of Multiparametric Flow Cytometry in CAR-T Immunotherapy

“…The previous category includes tumor cell immunophenotyping to select promising targets [17,18], minimal residual/measurable diseases (MRD) detection [3,19,20], and recovery kinetics of target antigen-positive cells [3, 21,22]. The latter category includes lymphocyte activity and function evaluation, CAR-positive cell assay [21][22][23][24][25], immune cell subsets detection [21][22][23][24][25][26][27][28][29][30][31], lymphocyte killing function assay [27], cytokines detection [26][27][28][29], tumor microenvironment (TME) evaluation, and immunosuppressive signals detection [23,26].…”

“…Multiparametric flow cytometry (MFC) is an immunological technique developed in the 1970s and has become an indispensable methodology for clinical diagnosis and basic research [15,16]. CAR-T is one kind of immunotherapy, and MFC plays a pivotal role in the entire process of CAR-T [3,[17][18][19][20][21][22][23][24][25][26][27][28][29]. These applications involve multiple aspects of MFC, and basically can be divided into two categories, that is, protocols Above and optionally TME and immune checkpoint.…”