Molecular determinants of chaperone interactions on MHC-I for folding and antigen repertoire selection.

The interplay between a highly polymorphic set of MHC-I alleles and molecular chaperones shapes the repertoire of peptide antigens displayed on the cell surface for T cell surveillance. Here, we demonstrate that the molecular chaperone TAP-binding protein related (TAPBPR) associates with a broad range of partially folded MHC-I species inside the cell. Bimolecular fluorescence complementation and deep mutational scanning reveal that TAPBPR recognition is polarized toward the α2 domain of the peptide-binding groove, and depends on the formation of a conserved MHC-I disulfide epitope in the α2 domain. Conversely, thermodynamic measurements of TAPBPR binding for a representative set of properly conformed, peptide-loaded molecules suggest a narrower MHC-I specificity range. Using solution NMR, we find that the extent of dynamics at "hotspot" surfaces confers TAPBPR recognition of a sparsely populated MHC-I state attained through a global conformational change. Consistently, restriction of MHC-I groove plasticity through the introduction of a disulfide bond between the α1/α2 helices abrogates TAPBPR binding, both in solution and on a cellular membrane, while intracellular binding is tolerant of many destabilizing MHC-I substitutions. Our data support parallel TAPBPR functions of 1) chaperoning unstable MHC-I molecules with broad allele-specificity at early stages of their folding process, and 2) editing the peptide cargo of properly conformed MHC-I molecules en route to the surface, which demonstrates a narrower specificity. Our results suggest that TAPBPR exploits localized structural adaptations, both near and distant to the peptide-binding groove, to selectively recognize discrete conformational states sampled by MHC-I alleles, toward editing the repertoire of displayed antigens

The APPLe Study: A Randomized, Community-Based, Placebo-Controlled Trial of Azithromycin for the Prevention of Preterm Birth, with Meta-Analysis

In a randomized trial in Malawi of azithromycin versus placebo in over 2,000 pregnant women, Jim Neilson and colleagues show no benefit of azithromycin for a number of outcomes including preterm birth and prenatal death

c-Rel Controls Multiple Discrete Steps in the Thymic Development of Foxp3+ CD4 Regulatory T Cells

The development of natural Foxp3+ CD4 regulatory T cells (nTregs) proceeds via two steps that involve the initial antigen dependent generation of CD25+GITRhiFoxp3−CD4+ nTreg precursors followed by the cytokine induction of Foxp3. Using mutant mouse models that lack c-Rel, the critical NF-κB transcription factor required for nTreg differentiation, we establish that c-Rel regulates both of these developmental steps. c-Rel controls the generation of nTreg precursors via a haplo-insufficient mechanism, indicating that this step is highly sensitive to c-Rel levels. However, maintenance of c-Rel in an inactive state in nTreg precursors demonstrates that it is not required for a constitutive function in these cells. While the subsequent IL-2 induction of Foxp3 in nTreg precursors requires c-Rel, this developmental transition does not coincide with the nuclear expression of c-Rel. Collectively, our results support a model of nTreg differentiation in which c-Rel generates a permissive state for foxp3 transcription during the development of nTreg precursors that influences the subsequent IL-2 dependent induction of Foxp3 without a need for c-Rel reactivation

Self-antigen recognition, Treg cell development and autoimmune disease

© 2015 Dr. Sarah Anne OverallThe recognition of self-antigen by the T cell receptor (TCR) defines T cell biology, orchestrating Treg differentiation, T cell survival and the decision between tolerance and immunity. How TCR engagement influences thymocyte commitment to the Treg cell lineage and the size of the thymic Treg niche is ambiguous. It is also unclear how the recognition of self-antigen by the TCR contributes to auto-reactive T cell survival in the periphery of immunosufficient animals and how auto-reactive T cells responding to self-antigen in the periphery are tolerised during inflammatory responses. The requirement for self-antigen recognition in promoting Treg development is unequivocal and has been extensively studied. Nonetheless, we do not understand why different TCRs promote Treg induction to differing degrees or why only a small percentage of clonally identical thymocytes develop into Tregs even in TCR transgenic mice. Competition between clones for antigenic stimulation has been widely suggested to limit the induction of Tregs in the thymus, however, this model is inconsistent with monoclonal TCR transgenic studies in which increased antigen presentation does not enhance Treg frequency, suggesting additional factors such as cytokine availability may limit the Treg niche. Competition for cytokines, particularly interleukin-2 (IL-2) is thought to limit the thymic Treg niche by restricting the number of CD25+Foxp3- precursors which upregulated Foxp3. However, while the importance of IL-2 in Foxp3 upregulation in CD25+Foxp3- Treg precursors is unequivocal, there is little direct evidence to suggest that IL-2 availability specifically limits thymic Treg development. The detection of CD25-Foxp3+ cells (CD25-Tregs) in IL-2-/- mice indicates that IL-2 many not be essential for Foxp3 expression in these cells and thus may represent a developmentally unique subset of thymic Tregs distinct from classical CD25+Tregs. Downstream of TCR stimulation and cytokine receptor signaling is the molecule mTOR (molecular target of rapamycin), which plays an important role in providing survival signals and enhancing proliferative potential in thymocytes and mature T cells. In mature T cells, inhibition of mTOR with the drug rapamycin promotes Foxp3 expression. Furthermore, mTOR-/- T cells readily develop into Foxp3+Tregs following TCR stimulation raising the possibility that mTOR activity in thymocytes may limit commitment to the Treg lineage. However, little has been done to assess the effect of mTOR inhibition on thymic Treg development. The recognition of self-antigen in the periphery is also necessary for the induction of peripheral tolerance and the initiation of autoimmunity. In mouse models of autoimmune gastritis, highly pathogenic gastritogenic T cells are eliminated from polyclonal repertoires by peripheral deletion. However, gastritogenic T cells of lower pathogenicity are not deleted. The tolerance mechanisms which control the activation of these lower pathogenic T cells in immunosufficient hosts is unclear. Furthermore, the potential for these surviving gastritogenic T cells to become activated under inflammatory conditions, where both self and non self-antigens are presented, is unknown. This is of particular importance in the development of gastritis in which T cells which recognise the subunit of the gastric H+/K+ ATPase (HK) are not tolerised in the thymus. In this study I have used foetal thymic organ culture (FTOC) to investigate how the size of the Treg niche is influenced by the level of TCR stimulation, IL-2 availability and inhibition of mTOR by rapamycin. Using these systems I show that the size of the Treg niche is determined by the level of TCR stimulation and was not limited by antigen density. I found no evidence that IL-2 availability limits the size of the Treg niche in FTOC. In addition CD25-Tregs only require IL-2 for survival and not Foxp3 expression. CD25-Treg development was also promoted under conditions of reduced antigenic stimulation, collectively indicating that CD25-Tregs are developmentally distinct from CD25+Tregs. I also show that inhibition of mTOR by rapamycin potentiates Bim-mediated apoptosis of polyclonal Tregs in what appears to be a cell extrinsic mechanism. I also address the question of T cell tolerance to gastric antigens during inflammatory responses by using an adoptive transfer model in which cognate antigen is delivered by immunisation. I found that IE transgenic mice present gastric ATPase -subunit (HK) derived epitopes at an increased density in the paragastric lymph node and this increase in antigen presentation results in enhanced survival of HK-specific (A23) T cells rendering these mice susceptible to immunisation induced gastritis, whereas HK peptide immunised wild-type mice did not develop disease. Anergy appears to be important in preventing gastritis induction by transferred A23 T cells in wild-type mice as the anergic status of A23 T cells was reduced in immunised IE mice compared to A23 T cells transferred into immunised wild-type mice. This work demonstrates the importance of self-antigen recognition in defining the size of the regulatory T cell niche in the thymus and the size of the auto-reactive T cell population in the periphery and highlights the critical role TCR interactions play in shaping the fate of not only individual cells but the impact these decisions have on T cell populations as a whole and ultimately the decision between tolerance and immunity

Biomolecular Perturbations in In-Cell Dynamic Nuclear Polarization Experiments

In-cell DNP is a growing application of NMR to the study of biomolecular structure and function within intact cells. An important unresolved question for in-cell DNP spectroscopy is the integrity of cellular samples under the cryogenic conditions of DNP. Despite the rich literature around cryopreservation of cells in the fields of stem cell/embryonic cell therapeutics, cell line preservation and in cryo-EM applications, the effect of cryopreservation procedures on DNP parameters is unclear. In this report we investigate cell survival and apoptosis in the presence of cryopreserving agents and DNP radicals. We also assess the effects of these reagents on cellular enhancements. We show that the DNP radical AMUPol has no effect on membrane permeability and does not induce apoptosis. Furthermore, the standard aqueous glass forming reagent, comprised of 60/30/10 d8-glycerol/D2O/H2O (DNP juice), rapidly dehydrates cells and induces apoptosis prior to freezing, reducing structural integrity of the sample prior to DNP analysis. Preservation with d6-DMSO at 10% v/v provided similar DNP enhancements per √unit time compared to glycerol preservation with superior maintenance of cell size and membrane integrity prior to freezing. DMSO preservation also greatly enhanced post-thaw survival of cells slow-frozen at 1°C/min. We therefore demonstrate that in-cell DNP-NMR studies should be done with d6-DMSO as cryoprotectant and raise important considerations for the progression of in-cell DNP-NMR towards the goal of high quality structural studies.ISSN:2296-889

Insertion Depth Modulates Protein Kinase C-δ-C1b Domain Interactions with Membrane Cholesterol as Revealed by MD Simulations

Protein kinase C delta (PKC-δ) is an important signaling molecule in human cells that has both proapoptotic as well as antiapoptotic functions. These conflicting activities can be modulated by two classes of ligands, phorbol esters and bryostatins. Phorbol esters are known tumor promoters, while bryostatins have anti-cancer properties. This is despite both ligands binding to the C1b domain of PKC-δ (δC1b) with a similar affinity. The molecular mechanism behind this discrepancy in cellular effects remains unknown. Here, we have used molecular dynamics simulations to investigate the structure and intermolecular interactions of these ligands bound to δC1b with heterogeneous membranes. We observed clear interactions between the δC1b-phorbol complex and membrane cholesterol, primarily through the backbone amide of L250 and through the K256 side-chain amine. In contrast, the δC1b-bryostatin complex did not exhibit interactions with cholesterol. Topological maps of the membrane insertion depth of the δC1b-ligand complexes suggest that insertion depth can modulate δC1b interactions with cholesterol. The lack of cholesterol interactions suggests that bryostatin-bound δC1b may not readily translocate to cholesterol-rich domains within the plasma membrane, which could significantly alter the substrate specificity of PKC-δ compared to δC1b-phorbol complexes.ISSN:1422-006