Keichiro Mihara

Aggressive periodontitis causes rapid periodontal tissue destruction and is a disease that occurs at a young age and runs in the patient’s family. Here, we revealed a heterozygous A116V missense mutation in the gene encoding monocyte to macrophage differentiation associated 2 (MMD2) protein in a Japanese family with aggressive periodontitis and neutropenia. Analyses of patients’ peripheral blood revealed a low number of neutrophils but abundant quantity of CD34 ⁺ hematopoietic stem and progenitor cells (HSPCs). Moreover, mutant Mmd2 mice showed severe alveolar bone loss and neutropenia. In patients and mutant Mmd2 mice, differentiation of HSPCs into granulocytes was also impeded, and their granulocytes were functionally impaired. Taken together, A116V mutation in MMD2 gene induced mild neutropenia and slightly limited the immune defense response. Our studies suggested that aggressive periodontitis in association with A116V MMD2 mutation constitutes a new immune system defect that belongs to the same spectrum of severe congenital neutropenia.

Chimeric antigen receptors against CD19 (anti-CD19-CAR) are widely recognized and used by not only researchers associated with immunology, molecular biology, and cell biology but also physicians to treat B-cell malignancies. Anti-CD19-CAR is currently clinically available as one of the therapeutic modalities for refractory acute B-cell-typed lymphoblastic leukemia (B-ALL) patients. However, to detect CAR on the cell surface and investigate the efficacy of CAR-T cells, there are numerous experimental modalities including flow cytometry, the Cr-releasing assay, immunoblot, and immunostaining. We have chosen several techniques, which are necessary and sufficient as well as reliable and reproducible to detect and assess the killing effect of CAR-T cells. Here, we describe protocols for basic experiments and procedures for the detection of CAR on transduced cells and in in vitro coculture experiments to assess cytotoxicity using CAR-T cells.

Patients with B cell lymphomas bearing MYC translocation combined with translocation involving other genes, such as BCL2, BCL3, or BCL6, defined as double-hit lymphoma (DHL), have a poor prognosis. Recent studies expanded the concept to include double-expressing lymphoma (DEL) that co-overexpresses MYC protein with either of those proteins. Accordingly, we defined cytogenetic DHL and DEL as primary DHL. An adoptive T cell immunotherapy with a chimeric antigen receptor (CAR) has been clinically shown to exhibit cytotoxicity in refractory neoplasias. We revealed the marked cytotoxicity of anti-CD19- and/or anti-CD38-CAR T cells against primary DHL cells from patients. CD19- and/or CD38-specific T cells were co-cultured with cytogenetic DHL (n = 3) or DEL (n = 2) cells from five patients for 3 days. We examined whether T cells retrovirally transduced with each vector showed cytotoxicity against DHL cells. Anti-CD19- and/or anti-CD38-CAR T cells were co-cultured with primary DHL cells at an E:T ratio of 1:2 for 3 days. Anti-CD19- and anti-CD38-CAR T cells completely abrogated these DHL cells, respectively. Anti-CD19-CAR T cells synergistically exerted collaborative cytotoxicity against these primary DHL cells with anti-CD38-CAR T cells. Therefore, refractory DHL cells can be efficiently abrogated by the clinical use of T cells with anti-CD19- and/or anti-CD38-CAR.

We reported that T cells with anti‐CD38‐chimeric antigen receptors (CAR) eliminated B‐cell lymphoma cells expressing CD38. To employ anti‐CD38‐CAR against acute myeloid leukemia (AML) blasts not expressing CD38, it is necessary to induce or increase the intensity of CD38 expression. A lactate dehydrogenase (LDH)‐releasing assay and flow cytometry showed that anti‐CD38‐CAR T cells were cytotoxic against AML lines (THP‐1 and CMK) expressing high CD38 levels (>99%), in time‐ and number of effector‐dependent manners. In other AML lines (KG1, U937 and HL60) partially expressing CD38, CD38⁺ AML cells were killed by CD38‐specific T cells, but CD38⁻ AML cells remained survived. Intriguingly, 10 nM all‐trans retinoic acid (ATRA) augmented CD38 expression in KG1, U937 and HL60 cells and primary leukemic cells from AML patients. Moreover, the withdrawal of ATRA from the medium decreased CD38 expression in AML cells. Killing effects of anti‐CD38‐CAR T cells against AML lines and AML cells were limited without ATRA, whereas CD38‐specific T cells enhanced cytotoxicity on AML cells by ATRA in association with enhanced CD38 expression. These results indicate that anti‐CD38‐CAR T cells eliminate AML cells through CD38 expression induced by ATRA.

The abnormal expression of microRNAs (miRNAs) is implicated in various human diseases, including cancers. Accordingly, miRNA expressions have been examined in many cancer tissues and blood, but there have been few studies examining smear samples from bone marrow (BM) or peripheral blood. Here we successfully isolated small RNAs from BM smears using a mirVana miRNA Isolation Kit with our original modifications. The isolated small RNAs were then used to measure the levels of representative miRNAs such as miR-155, let-7a, and U6 via real-time PCR with a specific TaqMan probe, although peaks for the ribosomal RNAs (18S, and 28S) were not identified. The PCR curves of the miRNAs were indistinguishable from those from BM living cells from the same donor. Finally, our method for BM smears identified numerous abnormally altered miRNAs (significantly decreased, 39 miRNAs; significantly increased, 27 miRNAs) in follicular lymphomas (FL) compared with normal donors via TaqMan real-time PCR miRNA array. The array indicated that miR-451 showed the greatest decrease in FL (a 345-fold decrease), while miR-338-5p showed the greatest increase in FL (172-fold) relative to normal donors. The miRNAs identified by our study might serve as markers to predict the invasion of FL cells into BM without biopsy. Furthermore, our method will provide a new avenue for the analysis of miRNAs in BM smear samples from various hematologic diseases.