Counting Antigen-Specific CD8 T Cells: A Reevaluation of Bystander Activation during Viral Infection

Abstract

Viral infections induce extensive T cell proliferation in vivo, but the specificity of the majority of the responding T cells has not been defined. To address this issue we used tetramers of MHC class I molecules containing viral peptides to directly visualize antigen-specific CD8 T cells during acute LCMV infection of mice. Based on tetramer binding and two sensitive assays measuring interferon-gamma production at the single-cell level, we found that 50%-70% of the activated CD8 T cells were LCMV specific [2 x 10(7) virus-specific cells/spleen]. Following viral clearance, antigen-specific CD8 T cell numbers dropped to 10(6) per spleen and were maintained at this level for the life of the mouse. Upon rechallenge with LCMV, there was rapid expansion of memory T cells, but after infection with the heterologous vaccinia virus there was no detectable change in the numbers of LCMV-specific memory CTL. Therefore, much of the CD8 T cell expansion seen during viral infection represents antigen-specific cells and warrants a revision of our current thinking on the size of the antiviral response.

Effector CD8 T cells dedifferentiate into long-lived memory cells

“…Effector and memory antigen-specific CD8 T cells were identified and purified by Fluorescence Activated Cell Sorting (FACS) using H-2D b tetramers bound to LCMV peptide GP33-41 conjugated to a fluorophore, along with CD8, CD44, CD127, Klrg1 and CD62L-fluorophore conjugated antibodies as previously described. 11,14,25,26 . To generate LCMV-specific CD8 T cell chimeras, transgenic P14 CD8 T cells with an engineered TCR that recognize the epitope GP33-41 of LCMV were harvested from naïve P14 TCR transgenic mice and adoptively transferred intravenously to C57BL/6 mice (cell# / mouse is specified in the figure legend) 11,27–29 .…”

“…To generate LCMV-specific CD8 T cell chimeras, transgenic P14 CD8 T cells with an engineered TCR that recognize the epitope GP33-41 of LCMV were harvested from naïve P14 TCR transgenic mice and adoptively transferred intravenously to C57BL/6 mice (cell# / mouse is specified in the figure legend) 11,27–29 . Congenically marked LCMV-specific CD8 T cells were sorted using fluorescently labeled CD90.1 (Thy1.1) and CD8 antibodies as previously described 14 . Naïve antigen-specific cells obtained from transgenic P14 mice 30 were used as an antigen-specific naïve control.…”

“…We hypothesized that this would be a de novo process based on the rapid methylation and because this methylation was retained even while the CD8 T cells were rapidly dividing (10-15 divisions) during the first week after infection 14 . We have previously shown increased expression of the de novo methyltransferase Dnmt3a upon CD8 T cell activation 15 and therefore tested whether it was required for methylation of the CD62L promoter.…”

The secretory IFN-γ response of single CD4 memory cells after activation on different antigen presenting cell types

“…Mice in the middle of their natural life span were used because their bone marrow yields higher cell numbers than young mice. Femurs were flushed with DMEM (Life Technologies, Rockville, MD), and cells were passed through a 70-μm cell strainer, washed 1x with DMEM and incubated in 0.83% NH 4 Cl to lyse erythrocytes. The cells were then incubated for 1h at 4° C with a cocktail of antibodies purified from supernatants of B hybridomas GK1.5 (anti-CD4), 53-6.72 (anti-CD8), RA3-3A1/61 (anti-B220), H116-32 (anti-I-A K ), and 10-3.6.2 (anti-10-3.6.2 (anti-I-A k ) (American Type Culture Collection (ATCC), Manassas, VA); each antibody was present at 20 μg/10 8 bone marrow cells.…”

“…The advent of the tetramer technology has represented a major step towards accomplishing this goal [3,4]. MHC-tetramers allow the identification of specific T cells by virtue of specific binding of MHC-peptide ligands to the T cell receptor (TCR).…”