Letter to the Editor

Patients with B cell lymphoma bearing MYC translocation combined with an additional translocation involving other genes, such as BCL2, BCL3, BCL6, or CCND1, whose category is defined as double-hit lymphoma (DHL), have a dismal prognosis [1]. Li et al. reported that the prognosis of B cell lymphoma patients expressing concurrent MYC and BCL2 proteins without translocations was also dismal as well as that of DHL-bearing translocations of MYC/BCL2 genes in terms of the prognosis [1,2,3,4]. Thus, recent studies have expanded the concept to include double-expressing lymphoma (DEL) that co-overexpresses MYC protein with those proteins. Accordingly, we defined cytogenetic DHL and DEL as primary DHL. An adoptive T cell immunotherapy with anti-CD19 chimeric antigen receptors (CAR) has been clinically shown to exhibit marked cytotoxicity in patients with relapsed and refractory B cell lymphoid neoplasias [5,6,7]. We also developed anti-CD38-CAR and demonstrated its marked cytotoxicity against various hematological malignancies [8, 9]. However, it has not been elucidated whether CAR therapy could be effective for patients with cytogenetic DHL and DEL. Here, we revealed the marked cytotoxicity of anti-CD19- and/or anti-CD38-CAR T cells as well as the synergy of both CARs against primary DHL cells.

Cytogenetic DHL (n = 3) or DEL (n = 2) cells of the lymph nodes were collected from five patients (Table 1) after obtaining appropriate informed consent. CD19+ CD20+ lymphoma cells accounted for over 90% (90–97%). DHL (DEL) cell line cells, bearing the translocation of the IgH/MYC gene as well as overexpression of BCL2 protein (KPUM-UH1) or these primary cells were cultured in RPMI-1640 complete medium.

Table 1 Patients’ profiles and cytotoxicity of T cells expressing anti-CD19- or anti-CD38-CAR against primary DHL cells
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The cutoffs for immunohistochemical positivity for BCL2, BCL6, and MYC (Abcam, Cambridge, MA, USA) were 50, 30, and 40% of microscopically observed lymphoma cells, respectively. FISH analyses were performed by SRL (Tokyo, Japan).

The retroviral vector of anti-CD19- and anti-CD38-CAR was previously developed [8,9,10]. To produce a RD114-pseudotyped retrovirus, MSCV-IRES-EGFP-anti-CD19-CAR or MSCV-IRES-EGFP-anti-CD38-CAR, pEQ-PAM3(-E), and pRDF were used to co-transfect 293T cells with Lipofectamine plus (Invitrogen, Carlsbad, CA, USA). Peripheral blood mononuclear cells of donors were cultured for 48 h with 7 μg/ml PHA-M (Sigma, St Louis, MO, USA), 200 IU/ml interleukin-2 (PeproTech, London, UK) in the complete medium as described previously [8,9,10]. These T cells were retrovirally transduced in the presence of 4 μg/ml polybrene (Sigma) in a retronectin-coated tube (Takara-Bio, Otsu, Japan). For the transduction of anti-CD38-CAR, an anti-CD38 antibody (CPK-H; MBL, Nagoya, Japan) was added to the culture medium to protect transduced T cells from autolysis through cross-linkage of the anti-CD38-CAR with intrinsic CD38 [8, 9]. For the subsequent co-culture experiments, transduced T cells expressing green fluorescent protein (GFP) were sorted by FACSAria (BD). The specimens from patients and donors were used after approval by the institutional review board of Hiroshima University.

Primary DHL cells co-cultured with anti-CD19- and/or anti-CD38-CAR T cells were harvested and stained with an anti-CD19 antibody-PE and anti-CD38 antibody-APC (BD). These cells were then analyzed by a flow cytometer. Specific cytotoxicity of anti-CD19- and/or anti-CD38-CAR T cells against CD19+ primary DHL cells was evaluated using the formula (B-A)/B, where A is the number of CD19+ GFP− cells or CD38+ GFP− cells after incubation with anti-CD19- or anti-CD38-CAR-expressing T cells, respectively, and B is the number of CD19+ GFP− or CD38+ GFP− cells after incubation with vector-transduced T cells [8,9,10].

We initially detected cytogenetic DHL and DEL (Additional file 1: Figure S1 and Table 1). Next, we confirmed that goat anti-mouse-IgG-PerCP, which cross-reacts with CAR and GFP of the vector, were co-expressed as an internal control in T cells retrovirally transduced (transduction efficiency: 67.42 ± 14.43% (n = 5) for anti-CD19-CAR, 63.21 ± 10.89% (n = 5) for anti-CD38-CAR).

Prior to co-culture experiments, we examined whether CD19+ primary DHL cells expressed CD38. We showed that >97% of DHL cells obtained from five patients expressed CD38 (Table 1). DHL (DEL) cell line cells (KPUM-UH1) expressing CD19 and CD38 were co-cultured with anti-CD19- or anti-CD38-CAR T cells at an effector (E) target (T) ratio of 1:2 for 3 days. Co-culture experiments showed that either anti-CD19- or anti-CD38-CAR T cells almost completely eradicated KPUM-UH1 cells (Fig. 1a). As further experiments, CD19- or CD38-specific T cells were co-cultured with cytogenetic DHL (n = 3) or DEL (n = 2) cells from five patients at an E:T ratio of 1:2 for 3 days. Similarly, anti-CD19- or anti-CD38-CAR T cells completely abolished the primary DHL cells, respectively (Fig. 1a, c and Table 1). Using DHL cells from patient 2, we confirmed that each of the CAR T cells eliminated DHL cells in a cell-number-dependent manner (Fig. 1b, c). Additionally, anti-CD19-CAR T cells synergistically exerted a collaborative cytotoxicity against DHL cells from patient 2 with anti-CD38-CAR T cells, as shown in Fig. 1c. The simultaneous combination index was less than 1.0, leading to the synergy according to Calcusyn software (Biosoft, Cambridge, UK).

Fig. 1
figure 1

Cytotoxic effect of T cells with anti-CD19- and/or anti-CD38-CAR against DHL cells. a KPUM-UH1(DHL cell line) cells were co-cultured with mock, anti-CD19-, or anti-CD38-CAR T cells at an E:T ratio of 1:2 for 3 days. The cells were harvested and stained with an anti-CD38 antibody-APC and anti-CD19 antibody-PE. These cells were then analyzed by a flow cytometer. Anti-CD19- or anti-CD38-CAR T cells killed KPUM-UH1 cells, respectively (upper panels). Primary DHL cells from patients (patients 4 (cytogenetic DHL) and 5 (DEL)) were co-cultured with either of mock, anti-CD19-, or anti-CD38-CAR T cells at an E:T ratio of 1:2 for 3 days. Anti-CD19- or anti-CD38-CAR T cells eliminated primary DHL cells, respectively (middle and lower panels). The viable primary DHL cell population is indicated by the arrowhead. b Cytogenetic DHL cells from patient 2 (1 × 105 cells) were co-cultured with anti-CD19- or anti-CD38-CAR T cells for 3 days at various ratios to effector cells (0.5 × 105, 0.25 × 105, 0.05 × 105, and 0.025 × 105 cells). Each type of CAR T cells abrogated cytogenetic DHL cells in a cell-number-dependent manner. The viable cytogenetic DHL cell population is indicated by the arrowhead. c The specific cytotoxic effect of anti-CD19- and/or anti-CD38-CAR transduced T cells against DHL cells was cell-number-dependent

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These results showed that primary DHL cells, which are refractory or resistant to existing chemotherapeutic agents, can be efficiently abrogated by the clinical use of T cells with anti-CD19- and/or anti-CD38-CAR. Taken together, these results may warrant adoptive immunotherapy with T cells transduced with anti-CD19- and/or anti-CD38-CAR for patients with refractory cytogenetic DHL and DEL.