There is ongoing interest in using antigen-specific CD8 cells induced by vaccines to treat infectious, neoplastic, and transplantation-related diseases. One major obstacle has been difficulty in generating and maintaining strong CD8 responses in vivo. To dissect the relationship between antigen presentation and antigen-specific CD8 T cell responses, my lab has been studying the murine response to the pathogenic bacterium Listeria monocytogenes (LM), and especially to the LM product, lemA. LemA contains a highly immunogenic aminoterminal fragment, f-MIGWII, which can be presented to CD8 cells by the MHC class Ib product H2M3. In prior years, I have cloned and purified a recombinant form of lemA (r-lemA), confirmed that it can be effectively processed and presented to CD8 T cells in vitro, and demonstrated that lemA-treated mice generate large numbers of f-MIGWII-specific CD8 T cell in response to inoculation. In our more recent studies, we use lemA as a tool to study immune CD8 T cell generation and function. To assess the protective potential of lemA-immune cells in vivo, during the past year we have examined host resistance against LM in lemA-treated animals. We found that inoculated mice have markedly increased antilisterial immunity 7 days post inoculation, but do not retain this resistance one month later, even though they still have substantial numbers of lemA-immune CD8 memory cells. The reasons for this discrepancy remain as yet unclear. These studies demonstrate that some subsets of microbe-specific CD8 cells have surprisingly selective roles in the host defense. In this case, f-MIGWII-immune cells appear to contribute significantly to the containment of primary disease but not in the maintenance of long-term memory. Prospective screening for such selectivity in function may be a valuable precaution whenever potential immunogens are being considered for possible use in antimicrobial vaccines. To examine the impact of excess free immunogenic peptides on CD8 T cell responses, we have inoculated mice with mixtures of lemA and f-MIGWII, the active immunogenic peptide recognized by lemA-immune CD8 cells. Although infusions of f-MIGWII alone do not produce measurable responses, mice inoculated with an equimolar mixture of f-MIGWII and lemA demonstrate only 1/3rd the expected normal lemA-immune response, and the lemA immune cells generated show reduced avidity for f-MIGWII in vitro. These findings have several practical implications. First, they demonstrate that overly aggressive use of immunogenic antigens in vivo can adversely affect immune CD8 responses. Conversely, these studies suggest short term, high dose peptide treatment at the time of immune stimulation may be a useful approach for selectively downregulating subsets of antigen-specific effectors in vivo. Such a strategy may be valuable, for example in probing the physiologic role of epitope specific CD8 subsets in host defense against pathogens. H2M3-restricted lemA-immune T cells are an atypical CD8 subpopulation. To study lemA-like products and other antigens in a more conventional model system, we have generated a lemA-like construct (lemS) which contains a model immunogenic peptide Ova257-264, derived from ovalbumin, inserted immediately adjacent to the backbone of lemA. The resulting construct is processed by APC with the release of Ova257-264, an immunogenic peptide which is readily presented to CD8 cells by the class Ia MHC product H2Kb. Using a series of recently published assays and reagents (including MHC-peptide tetramers) we have compared antigen presentation by CD8 T cells in the days immediately after inoculation with the ovalbumin, ovalbumin in incomplete Freund's adjuvant (IFA), and lemS, and assessed the ultimate CD8 response to inoculation 7 days later. At equal doses ovalbumin, infused subcutaneously in vivo produces a different pattern of antigen presentation and CD8 T cell response than ovalbumin in incomplete Freund's adjuvant (IFA), and lemS. The ova257-264 element in ovalbumin is presented in vivo quite strongly. By contrast, Ova257-264 presentation is barely detectable after inoculation with ova/IFA or lemS. Despite lower antigen presentation, ovalbumin/IFA and lemS each elicit 5-10 fold greater CD8 responses in vivo than soluble ovalbumin, demonstrating clearly that even very low levels of antigen presentation can be sufficient to stimulate an extensive CD8 response. On the other hand, extensive antigen presentation alone clearly is not sufficient to assure a brisk CD8 response. Presumably concurrent """"""""costimulation"""""""" (provided for example by IFA or the lemA7-33 backbone) must be an important factor in explaining the differences in CD8 responses. In addition, we speculate the failure of ovalbumin to stimulate a CD8 response could reflect in part active suppression of potential by the relatively large concentrations of immunogenic peptide generated in vivo. If so, the ability of IFA or the lemA7-33 backbone to prevent excessive antigen presentation may be a significant factor in explaining their adjuvant activity in vivo. During the coming year, we are planning to pursue studies in two areas. 1) Using the lemA model system, we will to examine the mechanisms responsible for the peptide-induced downregulation immune CD8 responses in vivo, and examine strategies for both maximizing and preventing this effect. The findings may have valuable implications both in vaccine development and in developing methods to induce CD8 T cell tolerance in vivo. 2) Using Ova257-264-based antigens, we plan to continue our studies of the relationship between antigen presentation and CD8 responses in vivo. Using active LM infection as a model, we will examine the interrelationship between """"""""costimulation"""""""", and antigenic peptide presentation in determining the fate of naive and immune CD8 T cells in vivo. These efforts hopefully will advance efforts to develop a more rational basis for vaccine-mediated manipulation of CD8 cell responses in vivo.
