Direct visualization of the dynamics of antigen presentation in human cells infected with cytomegalovirus revealed by antibodies mimicking TCR specificity.

There are no direct means to study class I MHC presentation in human normal or diseased cells. Using CMV-infected human cells and applying novel mAb that mimic T-cell receptor specificity directed toward the immunogenic epitope of the viral pp65 protein presented on HLA-A2 molecules, we directly imaged the dynamics of Ag presentation in infected cells. We demonstrate that following infection large intracellular pools of HLA-A2/pp65 complexes are localized to the Golgi. These HLA-A2/pp65 pools account for the majority of total HLA-A2 molecules in infected cells. Interestingly, these large pools are sequestered inside infected cells and only a small portion of them are exported to the cell surface. Virus-induced class I MHC down-regulation did not affect the intracellular pool of HLA-A2/pp65 complexes. Our data also suggest that proteasome function influences the release of class I complexes to the membrane. We present herein a new and direct molecular tool to study the dynamics of viral Ag presentation that may further elucidate the balance between immune response versus viral escape.

A novel postpriming regulatory check point of effector/memory T cells dictated through antigen density threshold-dependent anergy.

CTLs act as the effector arm of the cell-mediated immune system to kill undesirable cells. Two processes regulate these effector cells to prevent self reactivity: a thymic selection process that eliminates autoreactive clones and a multistage activation or priming process that endows them with a license to kill cognate target cells. Hitherto no subsequent regulatory restrictions have been ascribed for properly primed and activated CTLs that are licensed to kill. In this study we show that CTLs possess a novel postpriming regulatory mechanism(s) that influences the outcome of their encounter with cognate target cells. This mechanism gauges the degree of Ag density, whereupon reaching a certain threshold significant changes occur that induce anergy in the effector T cells. The biological consequences of this Ag-induced postpriming control includes alterations in the expression of cell surface molecules that control immunological synapse activity and cytokine profiles and induce retarded cell proliferation. Most profound is genome-wide microarray analysis that demonstrates changes in the expression of genes related to membrane potential, TCR signal transduction, energy metabolism, and cell cycle control. Thus, a discernible and unique gene expression signature for anergy as a response to high Ag density has been observed. Consequently, activated T cells possess properties of a self-referential sensory organ. These studies identify a new postpriming control mechanism of CTL with anergenic-like properties. This mechanism extends our understanding of the control of immune function and regulation such as peripheral tolerance, viral infections, antitumor immune responses, hypersensitivity, and autoimmunity.