Characterization of Pulmonary T Cell Response to Helper-Dependent Adenoviral Vectors following Intranasal Delivery

Abstract

In spite of the extensive research in the field of gene therapy, host immune responses continue to be the major barrier in translating basic research to clinical practice. Helper-dependent adenoviral (HD-Ad) vectors show great potential for pulmonary gene therapy, but the knowledge of pulmonary immune responses toward these vectors is very limited. In this study, we show that HD-Ad vectors are potent stimulators of dendritic cell (DC) maturation, thus leading to stimulation of T cell proliferation with ∼6% of naive CD4+ T cells from pulmonary mediastinal lymph node responding to HD-Ad-treated DCs. In contrast to the belief that HD-Ad vectors are unable to prime adaptive immune response, we show for the first time, through in vivo pulmonary studies in mice, that HD-Ad vectors can prime CD4+ and CD8+ T cell responses in the lung at high and substantially low doses. This indicates cross-presentation of HD-Ad-derived epitopes by DCs to prime CD8+ T cell responses. To assess the basis of pulmonary T cell response against HD-Ad vectors, we examined the response of conventional DCs (cDCs) and plasmacytoid DCs (pDCs) in the lung. In response to HD-Ad delivery, there is induction of maturation in both cDC and pDC subsets, but it is the cDCs, not pDCs, that migrate rapidly to draining lymph nodes within the first 2 days after vector delivery to prime adaptive immune response against these vectors. These findings have implications for development of strategies to prevent adaptive immune responses against gene therapy vectors.

Lessons learned from lung and liver in-vivo gene therapy: implications for the future

“…This reduction is the likely reason that transgene expression is more persistent than that observed for early rAd vectors, but as the reduction is not absolute the risks of immune system surveillance increase upon re-administration which might result in cessation of transgene expression [33]. Encouragingly, recent work has shown that HD-Ad can be used to successfully transduce respiratory tissue in mice, pigs and human airway cultures [34].…”

Uptake of apoptotic DC converts immature DC into tolerogenic DC that induce differentiation of Foxp3+ Treg

“…Bone marrow cells were isolated from tibia and femurs of adult mice and cultured in the presence of GM-CSF for 7 days as described previously [34]. Isolation of naïve CD4 1 CD25 -T cells Naïve CD4 1 CD25 -CD62L 1 T cells were isolated from spleens of mice using CD4 1 CD62L 1 naïve T-cell isolation kit in conjunction with MACS columns from Miltenyi Biotec (Auburn, CA), following the manufacturer's instructions.…”

“…Necrosis was induced by pelleting cells followed by three cycles of freeze and thaw. Similar protocol was used for the induction of splenocyte apoptosis, which was isolated from spleens of C57BL/6 mice as described previously [34].…”

“…Bone marrow cells were isolated from tibia and femurs of adult mice and cultured in the presence of GM-CSF for 7 days as described previously [34].…”

“…Necrosis was induced by pelleting cells followed by three cycles of freeze and thaw. Similar protocol was used for the induction of splenocyte apoptosis, which was isolated from spleens of C57BL/6 mice as described previously [34].Live DC and apoptotic DC/splenocytes or necrotic DC co-culture experiments Bone-marrow-derived immature live DC (100 000 cells/well) were co-cultured with apoptotic/necrotic DC or apoptotic splenocytes (1 000 000 cells/well). In some experiments, cytochalasin D (0.8 mg/ mL) was added to inhibit phagocytosis.…”