Does lineage plasticity enable escape from CAR-T cell therapy? Lessons from MLL-r leukemia.

The clinical success of engineered, CD19-directed chimeric antigen receptor (CAR) T cells in relapsed, refractory B-cell acute lymphoblastic leukemia (B-ALL) has generated great enthusiasm for the use of CAR T cells in patients with cytogenetics that portend a poor prognosis with conventional cytotoxic therapies. One such group includes infants and children with mixed lineage leukemia (MLL1, KMT2A) rearrangements (MLL-r), who fare much worse than patients with low- or standard-risk B-ALL. Although early clinical trials using CD19 CAR T cells for MLL-r B-ALL produced complete remission in most patients, relapse with CD19-negative disease was a common mechanism of treatment failure. Whereas CD19neg relapse has been observed across a broad spectrum of B-ALL patients treated with CD19-directed therapy, patients with MLL-r have manifested the emergence of AML, often clonally related to the B-ALL, suggesting that the inherent heterogeneity or lineage plasticity of MLL-r B-ALL may predispose patients to a myeloid relapse. Understanding the factors that enable and drive myeloid relapse may be important to devise strategies to improve durability of remissions. In this review, we summarize clinical observations to date with MLL-r B-ALL and generally discuss lineage plasticity as a mechanism of escape from immunotherapy.

Transposon-mediated generation of CAR-T cells shows efficient anti B-cell leukemia response after ex vivo expansion.

CAR-T-cell therapy has shown considerable advance in recent years, being approved by regulatory agencies in US, Europe, and Japan for the treatment of refractory patients with CD19+ B-cell leukemia or diffuse large B-cell lymphoma. Current methods for CAR-T-cell production use viral vectors for T-cell genetic modification and can take up to 15 days to generate the infusion product. The development of simple and less costly manufacturing protocols is needed in order to meet the increasing demand for this therapy. In this present work, we generated 19BBz CAR-T cells in 8 days using a protocol based on the non-viral transposon-based vector Sleeping Beauty. The expanded cells display mostly a central memory phenotype, expressing higher levels of inhibitory receptors when compared with mock cells. In addition, CAR-T cells were cytotoxic against CD19+ leukemia cells in vitro and improved overall survival rates of mice xenografted with human RS4;11 or Nalm-6 B-cell leukemias. Infused CAR-T cells persisted for up to 28 days, showing that they are capable of long-term persistence and antitumor response. Altogether, these results demonstrate the effectiveness of our protocol and pave the way for a broader application of CAR-T-cell therapy.

Zika virus infects human blood mononuclear cells.

BACKGROUND: Zika virus (ZIKV) infection gained public health concern after the 2015 outbreak in Brazil, when microcephaly rates increased in babies born from infected mothers. It was demonstrated that ZIKV causes a congenital Zika virus syndrome, including various alterations in the development of the central nervous system. Although the infection of cells from the nervous system has been well documented, less is known in respect of ZIKV ability to infect immune cells. Herein, we investigated if peripheral blood mononuclear cells (PBMCs), freshly-isolated from healthy donors, could be infected by ZIKV. METHODS: PBMCs from healthy donors were isolated and cultured in medium with ZIKV strain Rio-U1 (MOI = 0.1). Infection was analyzed by RT-qPCR and flow cytometry. RESULTS: We detected the ZIKV RNA in PBMCs from all donors by RT-qPCR analysis. The detection of viral antigens by flow cytometry revealed that PBMC from more than 50% the donors were infected by ZIKV, with CD3+CD4+ T cells, CD3-CD19+ B cells and CD3+CD8+ T cells being, respectively, the most frequently infected subpopulations, followed by CD14+ monocytes. Additionally, we observed high variability in PBMC infection rates among different donors, either by numbers or type infected cells. CONCLUSIONS: These findings raise the hypothesis that PBMCs can act as a reservoir of the virus, which may facilitate viral dissemination to different organs, including immune-privileged sites.

Maternal-Foetal Diabetes Modifies Neonatal Fc Receptor Expression on Human Leucocytes.

This study investigated the expression of the neonatal Fc receptor (FcRn) in maternal blood, cord blood and placental cells and determined IgG levels in maternal blood and cord blood from diabetic mothers. Peripheral blood, cord blood and placenta samples were collected from 26 mothers with normoglycaemia (non-diabetic, ND group) and 52 with hyperglycaemia (26 with mild gestational hyperglycaemia, MGH group, and 26 with type 2 diabetes mellitus, DM-2 group). Cells expressing CD19(+) and FcRn were identified by flow cytometry. Total IgG and its subclasses were quantified by ELISA. Maternal blood from DM-2 and cord blood from MGH exhibited a higher proportion of CD19(+) expression by B cells. DM-2 showed a lower proportion of CD19(+) cells in placenta. FcRn expression increased in cells from cord blood and placenta from MGH. Maternal blood, cord blood and placenta cells from DM-2 showed lower FcRn expression. Blood IgG levels were lower in DM-2, and cord blood IgG levels were higher in MGH. The highest levels of IgG4 were detected in the blood of hyperglycaemic mothers. The highest IgG3 and IgG4 levels in cord blood were detected in MGH, and the lowest IgG2 and IgG3 levels in DM-2. Maternal hyperglycaemia compromised placental transfer of IgG1, IgG3 and IgG4. The results suggest that regardless of hyperglycaemia degree, it decreases FcRn expression in placenta and blood cells and compromises the production and transfer of antibodies from maternal blood to newborns.

The CD19/CD81 complex physically interacts with CD38 but is not required to induce proliferation in mouse B lymphocytes.

In B lymphocytes, the cell surface receptor CD38 is involved in apoptosis of immature B cells, proliferation and differentiation of mature B cells. Although CD38 has been establish as a receptor, its signaling has been only partially characterized. As a result of the lack of signaling motifs in the cytoplasmic domain, CD38 must use a co-receptor to induce signaling within the cell. Accordingly, CD38 has been associated with different receptors such as the T-cell receptor/CD3 complex on T cells, CD16 on natural killer cells and MHC class II molecules on monocytes. The CD19/CD81 complex has been proposed as a co-receptor for CD38 in human B lymphocytes, but little or no characterization has been performed in mice. In this study the contribution of the CD19/CD81 complex in murine CD38 signaling was evaluated. Proliferation assays were performed using CD19(-/-) or CD81(-/-) deficient mice; CFSE-labeled B lymphocytes from wild-type mice and CD19(-/-) , CD81(-/-) and CD38(-/-) deficient mice were stimulated with agonistic antibodies against CD38. Immunoprecipitation and immunofluorescence were also performed to detect protein-protein interactions. Our results indicate that the CD19/CD81 complex interacts with CD38 but this interaction is not required to induce proliferation in mouse B lymphocytes, suggesting that other receptors may contribute to the proliferation induced by CD38 in B lymphocytes.

Individual and epistatic effects of genetic polymorphisms of B-cell co-stimulatory molecules on susceptibility to pemphigus foliaceus.

Following the candidate gene approach we analyzed the CD40L, CD40, BLYS and CD19 genes that participate of B-cell co-stimulation, for association with pemphigus foliaceus (PF), an organ-specific autoimmune disease, characterized by the detachment of epidermal cells from each other (acantholysis) and presence of autoantibodies specific for desmoglein 1 (dsg1), an epidermal cell-adhesion molecule. The disease is endemic in certain regions of Brazil and also is known as fogo selvagem. Complex interactions among environmental and genetic susceptibility factors contribute to the manifestation of this multifactorial disease. The sample included 179 patients and 317 controls. Strong significant association was found with CD40L-726T>C (odds ratio, OR=5.54 and 0.30 for T+ and C+ genotypes, respectively). In addition, there were significant negative associations with CD40 -1T (OR=0.61) and BLYS-871T (OR=0.62) due to the decrease of the frequency of both homo- and heterozygotes in the patient group. No associations were found with variants of CD19 gene. Gene-gene interactions were observed between CD40 and BLYS, and between CD40L and BLYS. So, the dominant protective effects of CD40L-726C and of CD40 -1T only manifest in BLYS-871T+ individuals, and vice versa. We conclude that genetic variability of CD40L, CD40 and BLYS is an important factor for PF pathogenesis.

Using cDNA microarrays to identify human CD19(+) B cell gene products (ESTs) originated from systemic lupus erythematosus susceptibility loci.

Systemic lupus erythematosus (SLE) is a prototype of autoimmune disease which arises from interactions between susceptibility genes and environmental factors. Despite the heterogeneous manifestations in this disease, all SLE patients present plasma autoantibodies recognizing nuclear components. Thus, auto reactive B cells represent key effectors to be investigated. Human linkage analysis is providing the localization of susceptibility loci distributed in chromosomes contributing to elucidate the manner in which interactions between these loci mediate SLE pathogenesis. We associate the cDNA microarray technology to investigate the differential gene expression of CD19(+) B cells with genetic linkage data. Bioinformatics programs served to evidentiate the differentially expressed sequences and the design of the microarray allowed hierarchical clustering of patients and controls. Sequencing allowed the identification of 8 new gene products differentially expressed (ESTs) that were co-localized in SLE or other autoimmune diseases susceptibility loci on chromosome 1p21, 2q21, 13q33, 16p12.1 and 16q12.1. These findings strongly suggest that chromosomal regions previously identified as SLE susceptibility loci are in fact transcribed in CD19(+) B cells of patients. In this review, we delineate a new possibility for the use of cDNA microarrays in studies focusing the control of gene expression of disease susceptibility loci identified by genetic linkage.