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Distinct Influences of Peptide-MHC Quality and Quantity on in Vivo T-Cell

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Distinct Influences of Peptide-MHC Quality and Quantity on in Vivo T-Cell Distinct influences of peptide-MHC quality and quantity on in vivo T-cell responses Rachel A. Gottschalka,b, Matthew M. Hathornb,c,1, Hélène Beuneud,1, Emily Corsea, Michael L. Dustind, Grégoire Altan-Bonnetc, and James P. Allisona,2 aDepartment of Immunology, Howard Hughes Medical Institute, and Ludwig Center for Cancer Immunotherapy, Memorial Sloan-Kettering Cancer Center, New York, NY 10065; bImmunology and Microbial Pathogenesis Program, Weill Cornell Graduate School of Medical Sciences, New York, NY 10021; cPrograms in Computational Biology and Immunology, Memorial Sloan-Kettering Cancer Center, New York, NY 10065; and dProgram in Molecular Pathogenesis, Helen L. and Martin S. Kimmel Center for Biology and Medicine of the Skirball Institute of Biomolecular Medicine, New York University School of Medicine, New York, NY 10016 Contributed by James P. Allison, December 9, 2011 (sent for review September 12, 2011) The strength of T-cell receptor (TCR) stimulation and subsequent strong pMHC). We used two previously characterized 5C.C7 T-cell response depend on a combination of peptide-major histo- TCR ligands presented in the context of I-Ek: moth cytochrome c compatibility complex (pMHC) density and potency. By comparing peptide 88–103 (MCC) (9) and 102S, a weak agonist peptide two different pMHC at doses yielding similar proliferation in vivo, resulting from an amino acid substitution at a single TCR contact we have highlighted unexpected differences in the qualitative and residue (10). The half-life of pMHC/TCR interactions is shorter quantitative effects of TCR ligand. Measurements of cytokine sen- for 102S than for MCC, as measured by either tetramer dissoci- – – sitivity and two-photon imaging of T cell dendritic cell (T DC) ation (t1/2 =33± 0.4 and 188.1 s, respectively) (8) or as calculated interactions reveal discrimination between comparably weak stim- from monomeric surface plasmon resonance measurements of uli resulting from either decreased pMHC potency or pMHC den- off-rate (t1/2 = 0.82 and 5.77 s, respectively) (11). sity. In addition, TCR-induced genes in broad gene expression Our results provide unique in vivo evidence for discrimination of profiles segregate into two groups: one that responds to cumula- pMHC potency, independent of the cumulative level of TCR signal, tive TCR signal and another that responds to pMHC quality, in- clearly demonstrating distinct influences of pMHC quality and dependent of quantity. These observations suggest that models quantity. Although pMHC density and potency appear interchange- IMMUNOLOGY of TCR ligand discrimination must account for disparate sensitivity able in the induction of proliferation in vivo, there are specific of downstream responses to specificinfluences of pMHC potency. effects of pMHC potency on the regulation of the IL-2 pathway. Furthermore, two-photon imaging indicates that T cells respond to affinity | IL-2 | STAT5 comparably weak low-potency and low-density stimuli in the context of distinct T cell–dendritic cell (T–DC) interactions. Finally, broad cells recognize peptide antigen in the context of MHC. gene expression patterns reveal a segregation of TCR-induced T During this process, the quality and quantity of the ligands responses into two groups: one that responds to the cumulative level determine the cumulative level of downstream signaling. Evi- of TCR signal and one that is sensitive to pMHC potency inde- dence suggests that T-cell receptor (TCR) can detect pMHC pendent of ligand density. Together these findings inform improved with remarkable sensitivity, responding to fewer than 10 pMHC models of TCR ligand discrimination and highlight the importance complexes (1, 2), and several models of TCR triggering have of distinguishing between pMHC density and potency during the been proposed to explain the ability of TCR to recognize various design of experimental systems and immunotherapies. pMHC ligands with such sensitivity, above the noise of abundant self-pMHC (3). Both TCR/pMHC affinity and off-rate can cor- Results relate with the potency of stimulation, depending on the on-rate of Peptide-MHC Density Can Compensate for Potency in the Induction the ligand (4, 5). Thus, the diverse nature of TCR/pMHC binding of Proliferation in Vivo. In vivo T-cell proliferation is influenced properties may limit the applicability of characterizing ligand by both pMHC density and potency (12). We addressed the re- quality on the basis of any single biochemical or thermodynamic lationship between these two variables in the initiation of pro- parameter. Here we use the term potency to more generally reflect liferation by titrating two previously characterized peptide ligands qualitative differences in TCR/pMHC binding parameters. for the 5C.C7 TCR. The natural ligand, MCC peptide, behaves as It is known from in vitro studies that the strength of TCR/ a strong agonist both in vitro and in vivo (8, 9), whereas 102S pMHC interactions sets dose requirements for initiation of a va- peptide, with both a lower affinity and a shorter half-life of TCR/ riety of T-cell responses (6). Higher peptide concentrations can pMHC–tetramer interactions, has a higher in vitro EC50 and results often compensate for weaker TCR/pMHC interactions to yield in decreased proliferation, cytokine production, and memory for- equivalent maximal responses in vitro. Thus, in studying the ef- mation in vivo (8, 10, 11). Both peptides bind comparably to MHC fects of altering the individual components of cumulative TCR (13) and are cleared at a similar rate in vivo (14). Consistent signal (i.e., the potency or density of antigen), it is difficult to with our previous work (14), in vivo titrations of stimulating peptide discriminate between distinct influences of TCR/pMHC binding demonstrated that pMHC potency shifts the threshold for pro- parameters and the impact of shifting the overall TCR signal. For liferation without altering the sharpness of the proliferative example, whereas recent in vivo studies have demonstrated that altered TCR ligands can induce large differences in the degree of proliferation, cytokine production and memory formation (7, 8), Author contributions: R.A.G., M.M.H., H.B., E.C., and G.A.-B. designed research; R.A.G., it is unclear whether the observed functional consequences are M.M.H. and H.B. performed research; R.A.G., M.M.H., H.B., E.C., M.L.D., G.A.-B., and J.P.A. specific to qualitative differences in these ligands or could be analyzed data; and R.A.G. and J.P.A. wrote the paper. similarly influenced by the amount of antigen. Only by manipu- The authors declare no conflict of interest. lating both variables within the same system is it possible to de- Freely available online through the PNAS open access option. termine whether T cells can discern specificinfluences of pMHC 1M.M.H. and H.B. contributed equally to this work. quality and quantity. Thus, we compared weak stimuli in vivo, 2To whom correspondence should be addressed. E-mail: [email protected]. resulting from either decreased pMHC potency (large quantities This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. of weak pMHC) or decreased pMHC density (small quantities of 1073/pnas.1119763109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1119763109 PNAS Early Edition | 1of6 Downloaded by guest on September 26, 2021 A C of 102S and MCC resulting in comparable proliferation also yielded similar Akt phosphorylation and induction of Foxp3 (14). After 48 h of in vivo stimulation, adoptively transferred 5C.C7 T cells were isolated and stained for the proliferation antigen ki67. We determined stimulation conditions, referred to here as “high-dose” (2 μg) 102S and “low-dose” (0.2 μg) MCC, which induced comparable proliferation, consistently resulting in ex- pression of ki67 in fewer than half of 5C.C7 T cells (Fig. 1 B and C). Using these conditions, we compared weak stimuli resulting from either reduced pMHC potency or reduced pMHC density. Peptide-MHC Potency Dissociates IL-2 Sensitivity, Receptor Expression, and Production from Cumulative TCR Signal. In seeking functional responses differentially regulated by TCR ligand quality and quantity, the IL-2 pathway was of particular interest in light of our previous work, which indicated differential survival induced by B low-density and low-potency pMHC (14). In addition, the IL-2 pathway has known antiapoptotic properties (15, 16) and is reg- ulated by a variety of factors downstream of the TCR (17). Thus, we assessed the effects of pMHC potency and density on TCR- induced IL-2 sensitivity. 5C.C7 T cells were isolated from lymph nodes (LNs) 48 h after injection of high- or low-dose MCC or 102S. The T cells were then stripped of bound cytokine and briefly treated with varying doses of IL-2, before fixing and staining for phosphorylated STAT5 as a measure of responsiveness to IL-2. Fig. 1. Peptide-MHC density can compensate for potency in the induction of Compared with 102S-stimulated 5C.C7, both doses of MCC proliferation in vivo. Adoptively transferred 5C.C7 T cells were isolated from LNs resulted in increased IL-2 sensitivity (Fig. 2). High-dose 102S in- 48 h after injection of (A) a 10-fold titration or (B and C)high(2μg) or low (0.2 duced some sensitivity to IL-2; however, the amplitude of this re- μg) doses of MCC or 102S peptide. (A) Percentage of cells that had divided was B fl C sponse was reduced compared with cells responding to low-dose assessed using dilution of CFSE. ( ) Representative ow plots and ( ) pooled MCC (Fig. 2B). Thus, compared with the proliferative response frequencies of ki67 expression. Shaded histograms represent the no-peptide control. Data are representative of at least three independent experiments. (Fig. 1), increased pMHC density was unable to compensate for the low potency of the 102S ligand in terms of responsiveness to IL-2.
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