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.

Improving T Cell Expansion with a Soft Touch.

Protein-coated microbeads provide a consistent approach for activating and expanding populations of T cells for immunotherapy but do not fully capture the properties of antigen presenting cells. In this report, we enhance T cell expansion by replacing the conventional, rigid bead with a mechanically soft elastomer. Polydimethylsiloxane (PDMS) was prepared in a microbead format and modified with activating antibodies to CD3 and CD28. A total of three different formulations of PDMS provided an extended proliferative phase in both CD4+-only and mixed CD4+-CD8+ T cell preparations. CD8+ T cells retained cytotoxic function, as measured by a set of biomarkers (perforin production, LAMP2 mobilization, and IFN-γ secretion) and an in vivo assay of targeted cell killing. Notably, PDMS beads presented a nanoscale polymer structure and higher rigidity than that associated with conventional bulk material. These data suggest T cells respond to this higher rigidity, indicating an unexpected effect of curing conditions. Together, these studies demonstrate that adopting mechanobiology ideas into the bead platform can provide new tools for T cell based immunotherapy.

Sexually-dimorphic expression of tyrosine hydroxylase immunoreactivity in the brain of a vocal teleost fish (Porichthys notatus).

Vocal communication has emerged as a powerful model for the study of neural mechanisms of social behavior. Modulatory neurochemicals postulated to play a central role in social behavior, related to motivation, arousal, incentive and reward, include the catecholamines, particularly dopamine and noradrenaline. Many questions remain regarding the functional mechanisms by which these modulators interact with sensory and motor systems. Here, we begin to address these questions in a model system for vocal and social behavior, the plainfin midshipman fish (Porichthys notatus). We mapped the distribution of immunoreactivity for the catecholamine-synthesizing enzyme tyrosine hydroxylase (TH) in the midshipman brain. The general pattern of TH(+) cell groups in midshipman appears to be highly conserved with other teleost fish, with a few exceptions, including the apparent absence of pretectal catecholamine cells. Many components of the midshipman vocal and auditory systems were innervated by TH(+) fibers and terminals, including portions of the subpallial area ventralis, the preoptic complex, and the anterior hypothalamus, the midbrain periaqueductal gray and torus semicircularis, several hindbrain auditory nuclei, and parts of the hindbrain vocal pattern generator. These areas thus represent potential sites for catecholamine modulation of vocal and/or auditory behavior. To begin to test functionally whether catecholamines modulate vocal social behaviors, we hypothesized that male and female midshipman, which are sexually dimorphic in both their vocal-motor repertoires and in their responses to hearing conspecific vocalizations, should exhibit sexually dimorphic expression of TH immunoreactivity in their vocal and/or auditory systems. We used quantitative immunohistochemical techniques to test this hypothesis across a number of brain areas. We found significantly higher levels of TH expression in male midshipman relative to females in the TH cell population in the paraventricular organ of the diencephalon and in the TH-innervated torus semicircularis, the main teleost midbrain auditory structure. The torus semicircularis has been implicated in sexually dimorphic behavioral responses to conspecific vocalizations. Our data thus support the general idea that catecholamines modulate vocal and auditory processing in midshipman, and the specific hypothesis that they shape sexually dimorphic auditory responses in the auditory midbrain.