BCR signaling is required for posttransplant lymphoproliferative disease in immunodeficient mice receiving human B cells.

Posttransplant lymphoproliferative disease (PTLD) is a major therapeutic challenge that has been difficult to study using human cells because of a lack of suitable models for mechanistic characterization. Here, we show that ex vivo-differentiated B cells isolated from a subset of healthy donors can elicit pathologies similar to PTLD when transferred into immunodeficient mice. The primary driver of PTLD-like pathologies were IgM-producing plasmablasts with Epstein-Barr virus (EBV) genomes that expressed genes commonly associated with EBV latency. We show that a small subset of EBV+ peripheral blood-derived B cells expressing self-reactive, nonmutated B cell receptors (BCRs) expand rapidly in culture in the absence of BCR stimulation. Furthermore, we found that in vitro and in vivo expansion of EBV+ plasmablasts required BCR signaling. Last, treatment of immunodeficient mice with the BCR pathway inhibitor, ibrutinib, delays onset of PTLD-like pathologies in vivo. These data have implications for the diagnosis and care of transplant recipients who are at risk of developing PTLD.

A lentiviral vector B cell gene therapy platform for the delivery of the anti-HIV-1 eCD4-Ig-knob-in-hole-reversed immunoadhesin.

Barriers to effective gene therapy for many diseases include the number of modified target cells required to achieve therapeutic outcomes and host immune responses to expressed therapeutic proteins. As long-lived cells specialized for protein secretion, antibody-secreting B cells are an attractive target for foreign protein expression in blood and tissue. To neutralize HIV-1, we developed a lentiviral vector (LV) gene therapy platform for delivery of the anti-HIV-1 immunoadhesin, eCD4-Ig, to B cells. The EµB29 enhancer/promoter in the LV limited gene expression in non-B cell lineages. By engineering a knob-in-hole-reversed (KiHR) modification in the CH3-Fc eCD4-Ig domain, we reduced interactions between eCD4-Ig and endogenous B cell immunoglobulin G proteins, which improved HIV-1 neutralization potency. Unlike previous approaches in non-lymphoid cells, eCD4-Ig-KiHR produced in B cells promoted HIV-1 neutralizing protection without requiring exogenous TPST2, a tyrosine sulfation enzyme required for eCD4-Ig-KiHR function. This finding indicated that B cell machinery is well suited to produce therapeutic proteins. Lastly, to overcome the inefficient transduction efficiency associated with VSV-G LV delivery to primary B cells, an optimized measles pseudotyped LV packaging methodology achieved up to 75% transduction efficiency. Overall, our findings support the utility of B cell gene therapy platforms for therapeutic protein delivery.

Ex vivo engineered human plasma cells exhibit robust protein secretion and long-term engraftment in vivo.

Due to their unique longevity and capacity to secrete high levels of protein, plasma B cells have the potential to be used as a cell therapy for protein replacement. Here, we show that ex vivo engineered human plasma cells exhibit single-cell RNA profiles, scanning electron micrograph ultrastructural features, and in vivo homing capacity of long-lived plasma cells. After transferring human plasma cells to immunodeficient mice in the presence of the human cytokines BAFF and IL-6, we observe increases in retention of plasma cells in the bone marrow, with engraftment exceeding a year. The most profound in vivo effects of human IL-6 are observed within 20 days of transfer and could be explained by decreased apoptosis in newly differentiated plasma cells. Collectively, these results show that ex vivo engineered and differentiated human plasma cells have the potential for long-lived in vivo protein secretion, which can be modeled in small animals.