Development of optimized cytotoxicity assays for assessing the antitumor potential of CAR-T cells.

CAR-T cells are T cells expressing a chimeric antigen receptor (CAR) rendering them capable of killing tumor cells after recognition of a target antigen. CD19 CAR-T cells have revolutionized the treatment of hematological malignancies. Their function is typically assessed by cytotoxicity assays using human allogeneic cell lines expressing the target antigen CD19 such as Nalm-6. However, an alloreactive reaction is observed with these cells, leading to a CD19-independent killing. To address this issue, we developed a fluorescence microscopy-based potency assay using murine target cells to provide an optimized cytotoxicity assay with enhanced specificity towards CD19. Murine NIH/3T3 (3T3) fibroblast-derived cell line and EL4 T-cell lymphoma-derived cell line were used as targets (no xenoreactivity was observed after coculture with human T cells). 3T3 and EL4 cells were engineered to express eGFP (enhanced Green Fluorescent Protein) and CD19 or CD22 using retroviral vectors. CD19 CAR-T cells and non-transduced (NT) control T cells were produced from several donors. After 4 h or 24 h, alloreactive cytotoxicity against CD19+ Nalm-6-GFP cells and CD19- Jurkat-GFP cells was observed with NT or CAR-T cells. In the same conditions, CAR-T but not NT cells specifically killed CD19+ but not CD19- 3T3-GFP or EL4-GFP cells. Both microscope- and flow cytometry-based assays revealed as sensitive as impedance-based assay. Using flow cytometry, we could further determine that CAR-T cells had mostly a stem cell-like memory phenotype after contact with EL4 target cells. Therefore, CD19+ 3T3-GFP or EL4-GFP cells and fluorescence microscopy- or flow cytometry-based assays provide convenient, sensitive and specific tools to evaluate CAR-T cell function with no alloreactivity.

A major role for CD4+ T cells in driving cytokine release syndrome during CAR T cell therapy.

Anti-CD19 chimeric antigen receptor (CAR) T cell therapy represents a breakthrough for the treatment of B cell malignancies. Yet, it can lead to severe adverse events, including cytokine release syndrome (CRS), which may require urgent clinical management. Whether interpatient variability in CAR T cell subsets contributes to CRS is unclear. Here, we show that CD4+ CAR T cells are the main drivers of CRS. Using an immunocompetent model of anti-CD19 CAR T cell therapy, we report that CD4+, but not CD8+, CAR T cells elicit physiological CRS-like manifestations associated with the release of inflammatory cytokines. In CAR T cell-treated patients, CRS occurrence and severity are significantly associated with high absolute values of CD4+ CAR T cells in the blood. CRS in mice occurs independently of CAR T cell-derived interferon γ (IFN-γ) but requires elevated tumor burden. Thus, adjusting the CD4:CD8 CAR T cell ratio to patient tumor load may help mitigate CAR T cell-associated toxicities.

Tumor-intrinsic sensitivity to the pro-apoptotic effects of IFN-γ is a major determinant of CD4+ CAR T-cell antitumor activity.

CD4+ T cells and CD4+ chimeric antigen receptor (CAR) T cells display highly variable antitumor activity in preclinical models and in patients; however, the mechanisms dictating how and when CD4+ T cells promote tumor regression are incompletely understood. With the help of functional intravital imaging, we report that interferon (IFN)-γ production but not perforin-mediated cytotoxicity was the dominant mechanism for tumor elimination by anti-CD19 CD4+ CAR T cells. Mechanistically, mouse or human CD4+ CAR T-cell-derived IFN-γ diffused extensively to act on tumor cells at distance selectively killing tumors sensitive to cytokine-induced apoptosis, including antigen-negative variants. In anti-CD19 CAR T-cell-treated patients exhibiting elevated CAR CD4:CD8 ratios, strong induction of serum IFN-γ was associated with increased survival. We propose that the sensitivity of tumor cells to the pro-apoptotic activity of IFN-γ is a major determinant of CD4+ CAR T-cell efficacy and may be considered to guide the use of CD4+ T cells during immunotherapy.

Humoral and cellular responses to SARS-CoV-2 BNT162b2 vaccination in allogeneic hematopoietic stem cell transplantation recipients.

Previous studies reporting the response to SARS-CoV-2 mRNA vaccination in alloHSCT recipients used serological and/or cellular assays, but no study has evaluated vaccine-induced neutralizing antibodies. We prospectively studied 28 alloHSCT recipients who received two BNT162b2 doses. Two patients groups were defined according to time from alloHSCT and immunosuppressive treatment, and had different baseline immunologic status. Study end-point was the evaluation of humoral and cellular responses one month after the second vaccine. All patients seroconverted. Anti-S IgG levels and neutralizing antibodies percentages were not significantly different between both groups. Using IFNγ ELISpot assay, five patients showed a strong increase, without correlation with the humoral response. Using flow cytometry lymphocyte proliferation assay, 14 patients exhibited responding T cells, without difference between both groups or correlation with anti-S IgG levels. A few low serological responders had a detectable CD4 + T cell proliferative response. This finding should be confirmed in a larger cohort.

[Immunomonitoring of patients treated with CAR-T cells for hematological malignancy: Guidelines from the CARTi group and the Francophone Society of Bone Marrow Transplantation and Cellular Therapy (SFGM-TC)]. / Suivi immunologique des patients traités par cellules CAR-T pour hémopathie maligne: recommandations du groupe CARTi et de la Société francophone de greffe de moelle et de thérapie cellulaire (SFGM-TC).

CAR-T cells represent a new anti-tumor immunotherapy which has shown its clinical efficacy in B-cell malignancies. The results of clinical trials carried out in this context have shown that certain immunological characteristics of patients before (at the time of apheresis) and after the administration of the treatment, or of the CAR-T cells themselves, are correlated with the response to the treatment or to its toxicity. However, to date, there are no recommendations on the immunological monitoring of patients treated in real life. The objectives of this workshop were to determine, based on data from the literature and the experience of the centers, the immunological analyses to be carried out in patients treated with CAR-T cells. The recommendations relate to the characterization of the patient's immune cells at the time of apheresis, the characterization of the injected CAR-T cells, as well as the monitoring of the CAR-T cells and other parameters of immune reconstitution in the patient after administration of the treatment. Harmonization of practices will allow clinical-biological correlation studies to be carried out in patients treated in real life with the aim of identifying factors predictive of response and toxicity. Such data could have a major medico-economic impact by making it possible to identify the patients who will optimally benefit from these expensive treatments.

Inherited GATA2 Deficiency Is Dominant by Haploinsufficiency and Displays Incomplete Clinical Penetrance.

PURPOSE: Germline heterozygous mutations of GATA2 underlie a variety of hematological and clinical phenotypes. The genetic, immunological, and clinical features of GATA2-deficient patients with mycobacterial diseases in the familial context remain largely unknown. METHODS: We enrolled 15 GATA2 index cases referred for mycobacterial disease. We describe their genetic and clinical features including their relatives. RESULTS: We identified 12 heterozygous GATA2 mutations, two of which had not been reported. Eight of these mutations were loss-of-function, and four were hypomorphic. None was dominant-negative in vitro, and the GATA2 locus was found to be subject to purifying selection, strongly suggesting a mechanism of haploinsufficiency. Three relatives of index cases had mycobacterial disease and were also heterozygous, resulting in 18 patients in total. Mycobacterial infection was the first clinical manifestation in 11 patients, at a mean age of 22.5 years (range: 12 to 42 years). Most patients also suffered from other infections, monocytopenia, or myelodysplasia. Strikingly, the clinical penetrance was incomplete (32.9% by age 40 years), as 16 heterozygous relatives aged between 6 and 78 years, including 4 older than 60 years, were completely asymptomatic. CONCLUSION: Clinical penetrance for mycobacterial disease was found to be similar to other GATA2 deficiency-related manifestations. These observations suggest that other mechanisms contribute to the phenotypic expression of GATA2 deficiency. A diagnosis of autosomal dominant GATA2 deficiency should be considered in patients with mycobacterial infections and/or other GATA2 deficiency-related phenotypes at any age in life. Moreover, all direct relatives should be genotyped at the GATA2 locus.