In Vivo Cross-Presentation of a Soluble Protein Antigen: Kinetics, Distribution, and Generation of Effector CTL Recognizing Dominant and Subdominant Epitopes

Abstract

Cross-presentation of exogenous Ags via the MHC class I pathway is now recognized for its role in self-tolerance, tumor immunity, and vaccine development. However, little is known about the in vivo distribution and kinetics of cross-presented protein Ags, nor the subsequent development of CTL effector responses to dominant or subdominant epitopes. We examined the location and duration of cross-presented Ag by using 5,6-carboxy-succinimidyl-fluorescein ester-labeled T cells from class I-restricted Ag-specific TCR mice. Comparisons of results from an in vitro 51Cr release CTL assay with an in vivo CTL assay provided physiologically relevant insights into the functional capacities of CTL specific for epitopes with differing affinities. These data demonstrate that efficient cross-presentation of a dominant class I-restricted Ag is dose related and remains largely localized, but not limited to the draining lymph nodes for up to 3 wk following a single injection of soluble protein. Within this period, dominant peptide-specific CTL are fully functional in vivo throughout the secondary lymphoid system. However, no in vivo responses are seen to a subdominant or cryptic epitope. Prolonging Ag cross-presentation via use of IFA promoted persisting in vivo dominant epitope-specific CTL activity and revealed dose-responsive precursor CTL to the subdominant, but not to a cryptic epitope. Analysis of functional in vivo CTL responses demonstrated that, in the presence of strong ongoing responses to the dominant peptide, lytic activity of CTL directed at weaker epitopes is undetectable.

Involvement of cellular death in TRAIL/DR5-dependent suppression induced by CD4+CD25+ regulatory T cells

“…An in vivo killing assay was developed to measure in vivo evaluation of cytotoxic activity in the manner described by Chen et al 23 and Nelson et al 24 Activated Teff from BALB/c mice were labeled with either 0.5 or 5 mM CFSE (namely, CFSE low or CFSE high ). For i.v.…”

CRTAM Confers Late-Stage Activation of CD8+ T Cells to Regulate Retention within Lymph Node

“…Fourteen hours after injection, spleen cells were collected and CFSE-positive cells were analyzed by flow cytometry. NP-specific killing was calculated as follows: percentage of killing ϭ (1 Ϫ ((number of NP peptide-carrying cells (CFSE high ) in immunized mice)/(number of OVA peptide-carrying cells (CFSE low ) in immunized mice))/(number of CFSE high cells in normal mice)/(number of CFSE low cells in normal mice)) ϫ 100 (21).…”

Harnessing the immune response to treat cancer

“…Cross-presentation of tumour antigen bound to MHC class I is a constitutive feature during many types of tumour growth Robinson et al, 1999Robinson et al, , 2001) even in the absence of any anti-tumour CTL activity. Cell-bound antigen is presented more efficiently than soluble antigen (Li et al, 2001) and the degree of cross-presentation is dependent on the amount of antigen present (Nelson et al, 2000). Cross-presentation is dependent on TAP, and hence it was initially thought that for cross-presentation to occur, following cytosolic degradation by proteosomes, exogenous protein had to join the endogenous pathway in the endoplasmic reticulum (Brossart and Bevan, 1997).…”

“…Multiple tumour antigens generate a hierarchy of T-cell responses with dominant antigens producing stronger responses than sub-dominant or cryptic antigens (Wortzel et al, 1983;Nelson et al, 2000;Bundell et al, 2006). Tumour-specific neo-antigens are dominant over antigens shared with normal tissues (Lennerz et al, 2005).…”

“…Tumour-specific neo-antigens are dominant over antigens shared with normal tissues (Lennerz et al, 2005). CTL responses can be generated to weaker antigens, but require higher antigen concentrations and prolonged duration of exposure (Nelson et al, 2000). Furthermore, patients with differing human leucocyte antigen haplotypes may generate variable immune responses to the same tumour antigens or tumour vaccines because of differing affinity of the antigen for human leucocyte antigen molecules (Bioley et al, 2009b, c).…”