We have previously reported that the CD8+ T cell response elicited by recombinant adenovirus vaccination displayed a delayed contraction in the spleen. In our current study, we demonstrate that this unusual kinetic is a general phenomenon observed in multiple tissues. Phenotypic analysis of transgene-specific CD8+ T cells present 30 days postimmunization with recombinant adenovirus revealed a population with evidence of partial exhaustion, suggesting that the cells had been chronically exposed to Ag. Although Ag expression could no longer be detected 3 wk after immunization, examination of Ag presentation within the draining lymph nodes demonstrated that APCs were loaded with Ag peptide for at least 40 days postimmunization, suggesting that Ag remains available to the system for a prolonged period, although the exact source of this Ag remains to be determined. At 60 days postimmunization, the CD8+ T cell population continued to exhibit a phenotype consistent with partially exhausted effector memory cells. Nonetheless, these CD8+ T cells conferred sterilizing immunity against virus challenge 7–12 wk postimmunization, suggesting that robust protective immunity can be provided by CD8+ T cells with an exhausted phenotype. These data demonstrate that prolonged exposure to Ag may not necessarily impair protective immunity and prompt a re-evaluation of the impact of persistent exposure to Ag on T cell function.

Previous studies determined that the CD8؉ T-cell response elicited by recombinant adenovirus exhibited a protracted contraction phase that was associated with long-term presentation of antigen. To gain further insight into this process, a doxycycline-regulated adenovirus was constructed to enable controlled extinction of transgene expression in vivo. We investigated the impact of premature termination of transgene expression at various time points (day 3 to day 60) following immunization. When transgene expression was terminated before the maximum response had been attained, overall expansion was attenuated, yielding a small memory population. When transgene expression was terminated between day 13 and day 30, the memory population was not sustained, demonstrating that the early memory population was antigen dependent. Extinction of transgene expression at day 60 had no obvious impact on memory maintenance, indicating that maintenance of the memory population may ultimately become independent of transgene expression. Premature termination of antigen expression had significant but modest effects on the phenotype and cytokine profile of the memory population. These results offer new insights into the mechanisms of memory CD8 ؉ T-cell maintenance following immunization with a recombinant adenovirus.

We have recently reported that CD8 ؉ T-cell memory maintenance after immunization with recombinant human adenovirus type 5 (rHuAd5) is dependent upon persistent transgene expression beyond the peak of the response. In this report, we have further investigated the location and nature of the cell populations responsible for this sustained response. The draining lymph nodes were found to be important for primary expansion but not for memory maintenance, suggesting that antigen presentation through a nonlymphoid source was required. Using bone marrow chimeric mice, we determined that antigen presentation by nonhematopoietic antigenpresenting cells (APCs) was sufficient for maintenance of CD8 ؉ T-cell numbers. However, antigen presentation by this mechanism alone yielded a memory population that displayed alterations in phenotype, cytokine production and protective capacity, indicating that antigen presentation through both hematopoietic and nonhematopoietic APCs ultimately defines the memory CD8 ؉ T-cell response produced by rHuAd5. These results shed new light on the immunobiology of rHuAd5 vectors and provide evidence for a mechanism of CD8 ؉ T-cell expansion and memory maintenance that relies upon both hematopoietic and nonhematopoietic APCs. (Blood. 2011;117(4):1146-1155) IntroductionRecombinant adenovirus vectors have proven to be robust immunogens for eliciting T-cell immunity. 1-3 Vaccines derived from recombinant human adenovirus serotype 5 (rHuAd5) have displayed remarkable potency in various models prompting further investigation. To better understand the immunobiology of rHuAd5, we have been studying both the nature of the CD8 ϩ T cells elicited by these vaccines and the mechanisms of CD8 ϩ T-cell priming and memory maintenance. Immunization with rHuAd5 typically produces a sustained memory population with a protracted contraction phase, 4-8 although these kinetics may be influenced by vector configuration and route of administration. 9 The memory CD8 ϩ T-cell population is composed primarily of effector T cells (T EFF ) and effector memory T cells (T EM ), 5,6 which is indicative of a persistent viral infection. Indeed, we have recently determined that sustained, low-level antigen expression from the rHuAd5 vector plays a key role in maintaining the CD8 ϩ T-cell memory population. 10 Premature extinction of transgene expression causes pronounced CD8 ϩ T-cell contraction, but only modestly affects phenotype, suggesting that memory maintenance and phenotype may be regulated by distinct mechanisms.The relationship between memory CD8 ϩ T-cell phenotype and protective immunity remains to be fully established. T EM provide optimal immune protection against certain agents [11][12][13] ; therefore, understanding the nature of the antigen-presenting cells (APCs) involved in the generation and maintenance of CD8 ϩ T EM will provide important information for vaccine design. Dendritic cells (DCs) are thought to be critical for the induction of antiviral CD8 ϩ T-cell responses. At least 7 distinct DC populations have...

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We have investigated the role of CD4(+) T cells in the development of the CD8(+) T-cell response after immunization with recombinant adenovirus (rAd). In the absence of CD4(+) T cells, the "unhelped" CD8(+) T-cell population exhibited a reduction in primary expansion and long-term survival that appeared to be due to inadequate priming of naïve T cells. There were few functional or phenotypic differences between the helped and unhelped CD8(+) T-cell populations with the exception of O-glycosylated CD43, a marker of effector cells, which was augmented on the unhelped CD8(+) T-cell population. In some cases, the unhelped CD8(+) T-cell population exhibited reduced ability to control virus infection; however, this appeared to be a function of the reduced frequency of antigen-specific CD8(+) T cells. Most notably, the unhelped CD8(+) T-cell population exhibited no defect in secondary expansion. These results provide insight into the role of CD4(+) T cells during the primary CD8(+) T-cell response generated by rAd vaccines and identify potential benefits and issues that must be considered when using adenovirus vaccines under conditions where CD4(+) T-cell function may be limiting, such as vaccination of human immunodeficiency virus patients.