Interleukin-7 (IL-7) availability determines the size and proliferative state of the resting T cell pool. However, the mechanisms that regulate steady-state IL-7 amounts are unclear. Using experimental lymphopenic mouse models and IL-7-induced homeostatic proliferation to measure IL-7 availability in vivo, we found that radioresistant cells were the source of IL-7 for both CD4 and CD8 T cells. Hematopoietic lineage cells, although irrelevant as a source of IL-7, were primarily responsible for limiting IL-7 availability via their expression of IL-7R. Unexpectedly, innate lymphoid cells were found to have a potent influence on IL-7 amounts in the primary and secondary lymphoid tissues. These results demonstrate that IL-7 homeostasis is achieved through consumption by multiple subsets of innate and adaptive immune cells.

While a new generation of vaccine vectors has been developed for eliciting cellular immune responses, little is known about the optimal routes for their administration or about the ramifications of the kinetics of in vivo vaccine antigen expression for immunogenicity. We evaluated the kinetics of vaccine antigen expression by real-time in vivo photon imaging and showed dramatic differences in these kinetics using different vectors and different routes of administration. Further, using a gamma interferon enzyme-linked immunospot assay to measure T-lymphocyte immune responses, we observed an association between the kinetics of vaccine antigen expression in vivo and the magnitude of vaccine-elicited memory T-lymphocyte responses. These results highlight the utility of the real-time in vivo photon-imaging technology in evaluating novel immunization strategies and suggest an association between the kinetics of vaccine antigen clearance and the magnitude of vaccine-elicited T-lymphocyte memory immune responses.While a new generation of vaccine vectors has been created to induce cellular immune responses, we know little about how to use them to maximize the generation of memory T-lymphocyte populations. Recombinant viral vectors and plasmid DNA constructs have been shown to elicit potent cellular immune responses (2,7,8,13,15,16,21,23). However, little has been done to evaluate the optimal routes of administration of these vaccine modalities and the relationship between the route of administration and in vivo vaccine antigen expression. Moreover, we do not fully understand the ramifications of the kinetics of vaccine antigen expression for immunogenicity.The in vivo evaluation of new vaccine vectors to select optimal routes of administration, dose, and biodistribution has been difficult, requiring serial sacrifice of laboratory animals and assessment of individual organs for vaccine antigen expression. To simplify this process of vaccine evaluation, we have adopted an in vivo imaging system (IVIS) to measure the expression of luciferase by vaccine vectors. This imaging strategy harnesses the ability of the luciferase protein to catalyze the light-producing oxidation of the small molecule luciferin. Luciferin is inoculated into mice that have received luciferaseexpressing immunogens, and the quantity of light emitted by this reaction is monitored in living mice (6).The use of in vivo imaging and luciferase expression (4), a technology developed for evaluating gene therapy strategies, is well suited for studying these novel vaccine vectors. We have recently shown that IVIS can be used to monitor the distribution and kinetics of vaccine vectors in the living mouse (12). The present study was done to explore the impact of the route of vaccine administration on vaccine antigen expression. In the process of doing these studies, we observed a striking association between the kinetics of antigen expression in vivo and the induction of long-term memory T cells.

IntroductionPlasmid DNA vaccines are a promising modality for immunization against a variety of infectious agents because they are safe, readily scalable, and easy distributed. Plasmid DNA vaccine vectors can elicit CD8 ϩ cytotoxic T lymphocytes (CTLs), CD4 ϩ T helper cell immune responses, as well as humoral immune responses. Nonetheless, the utility of DNA immunogens has been limited by their failure to elicit sufficiently potent immune responses. 1 One potential explanation for the limited immunogenicity of plasmid DNA is that vaccine antigen expression is generated at only transient and at low levels. 1 Immune-mediated destruction of antigen-producing muscle fibers appears to play a significant role in limiting vaccine antigen expression. Clearance of antigen-expressing myocytes has been shown to be dependent both on the immunogenicity of the antigen and the presence of a functional immune system. 2,3 However, the cell types responsible for this destruction remain to be determined. We have shown that damping of plasmid DNA vaccine antigen expression in vivo occurs coincident with the emergence of major histocompatibility complex (MHC) class I-restricted T-cell responses. In addition, we observed that vaccine antigen expression persists in Fas receptor knockout mice, suggesting a role in this process for T cell-mediated apoptosis via the Fas/FasL pathway. 3 Based on these data, we hypothesized that CD8 ϩ T cells mediated vaccine antigen clearance through Fas-dependent apoptosis. Alternatively, other studies have suggested that the limited antigen expression in this setting may be a result of antibody-dependent cell-mediated cytotoxicity or complement-mediated lysis. 4 In addition to adaptive immune responses, innate immune responses, such as those mediated by macrophages and NK cells, have also been implicated as potential contributors to the destruction of antigenproducing myocytes. 5,6 In the present study, we investigated the cell types responsible for antigen clearance in plasmid DNA vaccinated mice. We used an In Vivo Imaging System (IVIS), which enabled us to measure antigen expression in vivo precisely, without serial killing of the animals. Using knockout (KO) mice and antibodydepletion experiments, we investigated the relative contribution of NK cells, macrophages, CD8 ϩ T cells, and CD4 ϩ T cells to the damping of antigen expression in vaccinated animals. Surprisingly, we observed that CD4 ϩ T cells were both necessary and sufficient to mediate plasmid DNA vaccine antigen clearance. These findings demonstrate a central role for CD4 ϩ T cells in vaccine antigen clearance. Methods Animals and immunizationsSix-to 8-week-old wild-type C57BL/6, C57BL/6.␤2 M KO, C57BL/ 6.MHC II KO, Rag1 KO, and NK-function-deficient beige mice (C57BL/6-Lyst bg7-9 ) were purchased from The Jackson Laboratory (Bar Harbor, ME). All animals were housed and maintained in accordance with the Guide for the Care and Use of Laboratory Animals, 10 and all studies and procedures were reviewed and approved by the Institutional Animal...