2D Kinetic Analysis of TCR and CD8 Coreceptor for LCMV GP33 Epitopes

The LCMV GP33 CD8 epitope has long been one of the most widely used antigens in viral immunology. Of note, almost all of the in vitro analyses of CD8 T cell responses to this epitope make use of an altered peptide ligand (APL) in which the cysteine from the original 9-mer peptide (KAVYNFATC) is substituted by a methionine at position 41 (KAVYNFATM). In addition, it is possible that the antigen processed during natural LCMV infection is an 11-mer peptide (KAVYNFATCGI) rather than the widely used 9-mer. Although previous affinity measurements using purified proteins for these antigen variants revealed minimal differences, we applied highly sensitive two dimensional (2D) biophysical based techniques to further dissect TCR interaction with these closely related GP33 variants. The kinetic analyses of affinity provided by the 2D micropipette adhesion frequency assay (2D-MP) and bond lifetime under force analyzed using a biomembrane force probe (BFP) revealed significant differences between 41M, 41C and the 11-mer 41CGI antigen. We found a hierarchy in 2D affinity as 41M peptide displayed augmented TCR 2D affinity compared to 41C and 41CGI. These differences were also maintained in the presence of CD8 coreceptor and when analysis of total TCR:pMHC and CD8:pMHC bonds were considered. Moreover, the three ligands displayed dramatic differences in the bond lifetimes generated under force, in particular the 41CGI variant with the lowest 2D affinity demonstrated a 15-fold synergistic contribution of the CD8 coreceptor to overall bond lifetime. Our analyses emphasize the sensitivity of single cell and single bond 2D kinetic measurements in distinguishing between related agonist peptides

Viral Escape Mutant Epitope Maintains TCR Affinity for Antigen yet Curtails CD8 T Cell Responses.

T cells have the remarkable ability to recognize antigen with great specificity and in turn mount an appropriate and robust immune response. Critical to this process is the initial T cell antigen recognition and subsequent signal transduction events. This antigen recognition can be modulated at the site of TCR interaction with peptide:major histocompatibility (pMHC) or peptide interaction with the MHC molecule. Both events could have a range of effects on T cell fate. Though responses to antigens that bind sub-optimally to TCR, known as altered peptide ligands (APL), have been studied extensively, the impact of disrupting antigen binding to MHC has been highlighted to a lesser extent and is usually considered to result in complete loss of epitope recognition. Here we present a model of viral evasion from CD8 T cell immuno-surveillance by a lymphocytic choriomeningitis virus (LCMV) escape mutant with an epitope for which TCR affinity for pMHC remains high but where the antigenic peptide binds sub optimally to MHC. Despite high TCR affinity for variant epitope, levels of interferon regulatory factor-4 (IRF4) are not sustained in response to the variant indicating differences in perceived TCR signal strength. The CD8+ T cell response to the variant epitope is characterized by early proliferation and up-regulation of activation markers. Interestingly, this response is not maintained and is characterized by a lack in IL-2 and IFNγ production, increased apoptosis and an abrogated glycolytic response. We show that disrupting the stability of peptide in MHC can effectively disrupt TCR signal strength despite unchanged affinity for TCR and can significantly impact the CD8+ T cell response to a viral escape mutant

Variant 35A stimulated cells demonstrate a decrease in metabolic fitness.

<p>P14 CD8+ T cells were assayed for glycolytic function using the Seahorse extracellular flux assay. (A) Glycolysis stress test assay was performed by sequentially adding the indicated reagents to purified CD8+ cells primed <i>in vitro</i> with either GP33 or 35A at day 3. Representative data shown (B) Graphical representation of stress test assay. * p < 0.05, **p < 0.01 (Two way ANOVA-Holm Sidak test). Data shown is the average of 8 experiments with samples run in quadruplicate. Glycolysis (C) and glycolytic capacity (D) were calculated from stress test ECAR values. Glycolysis was calculated by measuring the change in ECAR after addition of glucose and subtracting the basal ECAR levels. Glycolytic capacity is defined as difference between the maximum ECAR achieved after addition of oligomycin and basal ECAR. *p< 0.05, ^§p = 0.07 (Wilcoxon matched pairs signed rank test) Data shown is the average of 8 experiments with samples run in quadruplicate. (E) Glucose uptake was measured <i>ex vivo</i> from infected mice through the detection of fluorescent 2-NBDG by flow cytometry. Two independent experiments with 3 pooled mice in each experiment. Multiple t tests *p < 0.05 (F) <i>Ex vivo</i> cells were also analyzed for cell granularity (SSC) and size (FSC) using flow cytometry. Representative data shown. Error bars indicate ± SEM. Gray shaded histograms refer to cells isolated from uninfected control hosts or unstimulated cells.</p

CD25 expression, IL-2, and IFNγ are decreased after 35A stimulation despite equivalent early rounds of division.

<p>(A) CD25 expression was assayed on P14 T cells after 3 and 6 days of <i>in vitro</i> stimulation with 10μm peptide and correlated with division cycles indicated by CFSE. (B) and (C) Graphical representation of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149582#pone.0149582.g003" target="_blank">Fig 3A</a>. Average of 2 experiments. *p < 0.05 (Two way ANOVA-Holm Sidak). (D) IL-2 production after 24 and 72 hours of <i>in vitro</i> stimulation with peptide measured with ELISA. Average of 3–4 experiments with samples run in duplicate for each time point. (E) IFN<b>γ</b> production after 24 and 72 hours of in vitro stimulation with peptide measured via ELISA. Average of 2 experiments with samples run in duplicate for each time point. (F) Representative flow plot of <i>in vivo</i> IFN<b>γ</b> production at day 4 post infection. (G) Graphical representation of <i>in vivo</i> IFN<b>γ</b>. Average of 2 experiments with 3 mice pooled for each sample. *p = 0.0153 (Unpaired t test). Error bars indicate ± SEM.</p

Variant peptide 35A maintains the same affinity for TCR as native GP33 epitope despite MHC instability and decreased IRF4 expression.

<p>(A) Affinity of GP33 and 35A epitope using the two-dimensional (2D) micropipette adhesion system. Average of 5 independent experiments. Total of 43 pairs tested for GP33 and 41 pairs for 35A. p = 0.775 n.s. (two tailed unpaired T test) (B) Stability of peptide on MHC was measured using an RMAS assay by loading different concentrations of peptide on empty class I and assaying surface expression of H-2D^b after 30 minutes post loading. Average of two independent experiments. (C) 10μm of each peptide is loaded onto RMAS cells and the gMFI of H-2D^b is measured over time to assess peptide stability and half life. Average of 3 independent experiments. *p<0.05 (Two way ANOVA Holm-Sidak) (D) and (E) 10⁵ P14 CD8+ T cells were adoptively transferred into congenic hosts and infected with 2 x10³ PFU Clone 13 or 35A variant. At 4 and 8 days post infection, spleens were harvested analyzed by flow cytometry for IRF4 expression on donor cells. Average of 3–4 independent experiments with 3–4 mice pooled per group. *p<0.05 (Paired t test)(F) and (G) P14 CD8 T cells were stimulated <i>in vitro</i> with 10μm wildtype GP33 peptide or variant 35A peptide and harvested at indicated time points and analyzed by flow cytometry for IRF4 expression. Average of 3–4 independent experiments. *p<0.05 (Paired t test) (H) P14 T cells were stimulated with GP33 or 35A peptide over a range of doses and analyzed for IRF4 expression at day 3. Data shown is the average of 2 independent experiments. *p<0.05 (Multiple t tests) Gray shaded histograms refer to cells isolated from uninfected control hosts or unstimulated cells. Error bars on all plots indicate ± SEM</p

35A Stimulation results in reduced viability in responding cells.

<p>P14 CD8+T cells were stimulated with 10μm of GP33 or 35A peptide <i>in vitro</i> and evaluated for viability at day 3 and 6 post stimulation by staining with Annexin and 7AAD (A) or using Zombie Yellow cell viability dye (B). Representative flow plot shown in (A). Total of 4 experiments. ***p = 0.002 (Multiple T tests). Error bars indicate ± SEM. Cells were also assayed for the detection of pro-survival Bcl-2 molecule (C) Representative flow plot shown. Total of 2 experiments. Gray shaded histograms refer to unstimulated cells.</p

Recognition of 35A induces activation and proliferation of P14 CD8+ T cells without accumulation.

<p>(A) 10⁵ P14 Thy1.1 CD8+ T cells were adoptively transferred into C57BL/6 hosts and infected one day later with 2 x10³ PFU Clone 13 or 35A variant virus i.v. At 4 and 8 days post infection, spleens were harvested and analyzed for recovery of donor CD8+ T cells (A) and phenotypic analysis (B). Data from 3–5 experiments shown. n = 2–3 mice per group. *p<0.05 (2 way ANOVA multiple comparisons). Error bars indicate ± SEM. (C) P14 T cells were stimulated <i>in vitro</i> with 10μm GP33 or 35A peptide and harvested at day 3 or 6 to evaluate division by CFSE (C) total CD8+ T cell expansion (D) and phenotypic analysis (E). Representative data is shown in (C and E) or average of 3 experiments shown in (D) with samples run in duplicate for total cell number counts. *p<0.05 (2 way ANOVA multiple comparisons). Error bars indicate ± SEM. Gray shaded histograms refer to cells isolated from uninfected control hosts or unstimulated cells.</p