PD-1 signaling promotes control of chronic viral infection by restricting type-I-interferon-mediated tissue damage

Immune responses are essential for pathogen elimination but also cause tissue damage, leading to disease or death. However, it is unclear how the host immune system balances control of infection and protection from the collateral tissue damage. Here, we show that PD-1-mediated restriction of immune responses is essential for durable control of chronic LCMV infection in mice. In contrast to responses in the chronic phase, PD-1 blockade in the subacute phase of infection paradoxically results in viral persistence. This effect is associated with damage to lymphoid architecture and subsequently decreases adaptive immune responses. Moreover, this tissue damage is type I interferon dependent, as sequential blockade of the interferon receptor and PD-1 pathways prevents immunopathology and enhances control of infection. We conclude that PD-1-mediated suppression is required as an immunoregulatory mechanism for sustained responses to chronic viral infection by antagonizing type-I interferon-dependent immunopathology

Epigenetic and Signaling Pathways Regulating the Maintenance of CD8 T Cell Identity and Function

In response to infection, antigen specific CD4 and CD8 T cells rapidly divide to provide help to the immune system and promote cytotoxicity of infected cells, respectively. Through this rapid division, CD4 and CD8 T cells maintain silencing of the opposing lineage’s genes, which is essential to acutely eliminating pathogens. However, not all pathogens are acutely eliminated even when silencing is maintained, and the pathogen persists in the presence of activated CD8 T cells. CD8 T cells chronically exposed to antigen are phenotypically different than CD8 T cells acutely exposed to antigen, but CD8 T cell still exert control over chronic infections and cancers. Two unanswered questions regarding the maintenance CD8 T cell responses are: 1. How do CD8 T cells maintain the silencing of alternative lineage genes through division in the periphery, and 2. How do these cells maintain viral control through chronic stimulation. To shed light on these questions, two specific aims were developed for this thesis. The first specific aim was to determine whether the epigenetic factor G9a is required to maintain silencing of helper lineage genes in proliferating CD8 T cells. To this end, genetic deletion of G9a in CD8 T cells resulted in de-repression of Cd4 and other helper T-related genes during lymphopenia- or tumor antigen-induced proliferation. In response to Listeria monocytogenes infection, G9a deficient CD8 T cells maintained silencing of Cd4. These data highlight that proliferating CD8 T cells employ multiple gene silencing mechanisms including G9a–mediated epigenetic modifications to maintain silencing of T helper-associated genes. The second specific aim of this study was to determine how increasing PI3K signaling affects the maintenance of a functional CD8 T cell pool during chronic viral stimulation. During chronic Lymphocytic choriomeningtis virus (LCMV) infection, overexpression of a constitutively active form of PI3K in CD8 T cells caused lethal immunopathology reminiscent of chronic infection of PDL1 knockout mice. Inducible overexpression of PI3K after CD8 T cell priming depleted the memory- and stem-like CD8 T cell pool, which is required to sustain the CD8 T cell response. These data highlight an epistatic relationship between PI3K and PD1 in chronic CD8 T cells, and inhibitory signals may protect the chronic CD8 T cell progenitors from depletion throughout the course of infection. Future work will determine whether the responsiveness of CD8 T cells to PI3K signaling or PD1 blockade requires the transcription factor AP4

Role of Enthalpy–Entropy Compensation Interactions in Determining the Conformational Propensities of Amino Acid Residues in Unfolded Peptides.

The driving forces governing the unique and restricted conformational preferences of amino acid residues in the unfolded state are still not well understood. In this study, we experimentally determine the individual thermodynamic components underlying intrinsic conformational propensities of these residues. Thermodynamic analysis of ultraviolet-circular dichroism (UV-CD) and ¹H NMR data for a series of glycine capped amino acid residues (i.e., G-x-G peptides) reveals the existence of a nearly exact enthalpy–entropy compensation for the polyproline II−β strand equilibrium for all investigated residues. The respective Δ<i>H</i>_β, Δ<i>S</i>_β values exhibit a nearly perfect linear relationship with an apparent compensation temperature of 295 ± 2 K. Moreover, we identified iso-equilibrium points for two subsets of residues at 297 and 305 K. Thus, our data suggest that within this temperature regime, which is only slightly below physiological temperatures, the conformational ensembles of amino acid residues in the unfolded state differ solely with respect to their capability to adopt turn-like conformations. Such iso-equilibria are rarely observed, and their existence herein indicates a common physical origin behind conformational preferences, which we are able to assign to side-chain dependent backbone solvation. Conformational effects such as differences between the number of sterically allowed side chain rotamers can contribute to enthalpy and entropy but not to the Gibbs energy associated with conformational preferences. Interestingly, we found that alanine, aspartic acid, and threonine are the only residues which do not share these iso-equilbiria. The enthalpy–entropy compensation discovered as well as the iso-equilbrium and thermodynamics obtained for each amino acid residue provide a new and informative way of identifying the determinants of amino acid propensities in unfolded and disordered states

Ionized Trilysine: A Model System for Understanding the Nonrandom Structure of Poly-l-lysine and Lysine-Containing Motifs in Proteins

It is now well-established that different amino acid residues can exhibit different conformational distributions in the unfolded state of peptides and proteins. These conformational propensities can be modulated by nearest neighbors. In the current study, we combined vibrational and NMR spectroscopy to determine the conformational distributions of the central and C-terminal residues in trilysine peptides in aqueous solution. The study was motivated by earlier observations suggesting that interactions between ionized nearest neighbor residues can substantially change conformational propensities. We found that the central lysine residue predominantly adopts conformations that are located at the upper border of the upper left quadrant of the Ramachandran plot and the left border of the polyproline II region. We term this type of conformation deformed polyproline II (pPII^d). The structures of less populated subensembles of trilysine resemble are comparable with structures at the <i>i</i> + 1 position of type I and type II β-turns. For the C-terminal residue, however, we obtained a mixture of polyproline II, β-strand, and right-handed helical conformations, which is typical for lysine residues in alanine- and glycine-based peptides. Our data thus indicate that the terminal lysines modify and restrict the conformational distribution of the central lysine residue. DFT calculations for ionized trilysine and lysyllysyllysylglycine in vacuo indicate that the pPII^d is stabilized by a rather strong hydrogen bond between the NH₃⁺ group of the central lysine and the carbonyl group of the C-terminal peptide. This intramolecular hydrogen bonding induces optical activity in the C-terminal CO stretching vibration, which leads to an unusual and relatively intense positive Cotton band. Additionally, we analyzed the amide I′ band profile of ionized triornithine in water. Ornithine is structurally similar to lysine in that its side chain is terminated with an amino group; however, the side chain of ornithine is shorter than lysine’s side chain by one methylene group. We found that the conformational distribution of the central ornithine in this peptide must be very similar to that of the central lysine residue in trilysine. This suggests that the ionized ammonium group, which lysine and ornithine side chains have in common, is the main determinant of their conformational propensities at the central position in the respective tripeptides. The results of a DFT-based geometry optimization confirm this notion. In principle, our results suggest that lysine-rich segments in unfolded/disordered proteins and peptides can switch between different types of local order, i.e., an extended pPII^d-like conformation and transient turns. However, for longer polylysine segments nonlocal interactions between side chains might impede the formation of turns, thus enabling the formation of pPII^d-helix segments

pH-Independence of Trialanine and the Effects of Termini Blocking in Short Peptides: A Combined Vibrational, NMR, UVCD, and Molecular Dynamics Study

Several lines of evidence now well establish that unfolded peptides in general, and alanine in specific, have an intrinsic preference for the polyproline II (pPII) conformation. Investigation of local order in the unfolded state is, however, complicated by experimental limitations and the inherent dynamics of the system, which has in some cases yielded inconsistent results from different types of experiments. One method of studying these systems is the use of short model peptides, and specifically short alanine peptides, known for predominantly sampling pPII structure in aqueous solution. Recently, He et al. (J. Am. Chem. Soc. 2012, 134, 1571−1576) proposed that unblocked tripeptides may not be suitable models for studying conformational propensities in unfolded peptides due to the presence of end effect, that is, electrostatic interactions between investigated amino acid residues and terminal charges. To determine whether changing the protonation states of the N- and C-termini influence the conformational manifold of the central amino acid residue in tripeptides, we have examined the pH-dependence of unblocked trialanine and the conformational preferences of alanine in the alanine dipeptide. To this end, we measured and globally analyzed amide I′ band profiles and NMR J-coupling constants. We described conformational distributions as the superposition of two-dimensional Gaussian distributions assignable to specific subspaces of the Ramachandran plot. Results show that the conformational ensemble of trialanine as a whole, and the pPII content (χ_pPII = 0.84) in particular, remains practically unaffected by changing the protonation state. We found that compared to trialanine, the alanine dipeptide has slightly lower pPII content (χ_pPII = 0.74) and an ensemble more reminiscent of the unblocked Gly-Ala-Gly model peptide. In addition, a two-state thermodynamic analysis of the conformational sensitive Δε­(T) and ³<i>J</i>(H^NH^α)­(T) data obtained from electronic circular dichroism and H NMR spectra indicate that the free energy landscape of trialanine is similar in all protonation states. MD simulations for the investigated peptides corroborate this notion and show further that the hydration shell around unblocked trialanine is unaffected by the protonation/deprotonation of the C-terminal group. In contrast, the alanine dipeptide shows a reduced water density around the central residue as well as a less ordered hydration shell, which decreases the pPII propensity and reduces the lifetime of sampled conformations