Research in this laboratory is focused on achieving a mechanistic understanding of how the signals provided by antigen-presenting cells (APC) are integrated into the programmed response of CD8+ cytotoxic T lymphocytes (CTL). These studies are primarily based on the use of in-vivo animal models in which key parameters of the APC-CTL interaction can be manipulated and the result on the activation, expansion, function, and persistence of CTL can be studied. Specific goals include elucidating the instructional program that guides CTL development, defining the role of CD4+ T cells in providing ?ehelp?f to CTL, and understanding how APC participate in the regulation of CTL activation versus tolerance. It is anticipated that these studies will provide fundamental insights into biology of CTL responses and allow for their strategic manipulation in vaccine and immunotherapeutic settings.

Programming of CTL responses

We have developed a novel CTL priming system that allows precise control over the duration of antigenic stimulation provided to transgenic CTL as well as the selection of accompanying costimulatory and cytokine signals. This system features an adherent fibroblast APC engineered to express specific peptide/MHC class I complexes and secondary signals. Using this system, we have shown that a brief stimulation is sufficient to commit a nai?Nve CTL to subsequent clonal expansion and functional differentiation in vitro. As the inductive signals were received by the nai?Nve CTL prior to its first division, this subsequent response can be thought of having been guided by an instructional program that, once set into motion during priming, is executed without the need for further contact with APC. We have now extended these studies to investigate whether the duration of stimulation provided to a nai?Nve CTL can influence the extent and magnitude of its subsequent clonal expansion in vivo. We find that whereas CTL given a brief stimulation undergo an abortive clonal expansion in vivo prior to disappearing, CTL given a longer stimulation proceed through an extensive clonal expansion and, in contrast to briefly-stimulated CTL, are able to mediate destruction of peripheral target tissues. These disparate outcomes correlate with the ability of the CTL to sustain upregulation of the high-affinity interleukin-2 receptor, CD25. Taken together, these data indicate that the CTL program is not invariant, and is dynamic be modified or ?etuned?f to the level of antigenic stimulation provided. Our studies also demonstrate that the sensory apparatus used to initiate the developmental program of CTL can intergrate differences in signal strength into the decision to activate versus tolerize specific clones. Current efforts are directed towards understanding the intracellular pathways that are able to perceive differences in the strength or duration of antigenic signaling and the role of this mechanism in maintaining peripheral tolerance of T cells in vivo.

T-help for CTL

A long-standing paradox in cellular immunology concerns the requirement for CD4+ ?ehelper?f T cells in priming of CTL. Whereas CTL responses against certain viruses can occur in the absence of CD4+ cells, others, such as those mediated through ?ecross-priming?f by host APC are clearly dependent on the presence of CD4 T help. We have investigated the pathways through which T help is provided to CTL, as well as determining which aspects of CTL development are regulated by CD4 cells. One aspect of this work follows up on our previous finding that T help is communicated from CD4 T cells to APC via CD40L-CD40 interactions which leads to the activation of APC to a state in which they can autonomously prime CTL. We have now investigated how the ?ehelp message?f is transmitted by the activated APC to nai?Nve CTL. We have found that the upregulation of B7 molecules that accompanies APC activation is received as an increase in the signals transmitted through CD28 on CTL, leading to their primary activation. This represents a key step in CTL priming and shows that CD28 is not an ?eon-off?f switch, but rather can serve as a molecular rheostat able to integrate differences in the strength of signals it receives into the decision to prime CTL. We have also investigated which aspects of CTL development are influenced by T help using models of CD4-dependent and CD4-independent CTL responses. Our results show that T help is not required for the primary expansion and acquisition of effector function by CTL but is essential during priming to program the CTL with the capacity to undergo secondary expansion, a central hallmark of immune memory and protective immunity. Current efforts are directed towards defining the signals that direct primary versus secondary expansion, elucidating how ?ehelped?f CTL differ from ?ehelpless?f CTL, and determining whether physiological CTL responses contain CTL that are primed in the absence of T help.

CTL regulation by cross-presenting APC

One of the key functions assigned to APC is transport and cross-presentation of antigens obtained in the periphery to T cells in secondary lymphoid organs under the proper context such that the appropriate type of response can be induced. We have been investigating whether the form of antigenic encountered by cross-presenting APC can influence the nature of the subsequent response of CTL. We have found that, whereas introduction of a foreign model antigen expressed on a live or apoptotic cells results in immune tolerance, the same cells induced to undergo necrosis produces immune priming and memory development. CTL are induced to undergo primary expansion in either case, but when antigen derived from apoptotic cells is introduced, the CTL fail to develop important effector functions, such as cytokine secretion or cytotoxicity, that are intact when antigen is obtained from necrotic cells. Current efforts are directed towards understanding how APC are able to distinguish apoptotic from necrotic cells and how this information is intergrated into the specific signals provided to CTL which result in these disparate fates.