Presentation of an Immunodominant Immediate-Early CD8+ T Cell Epitope Resists Human Cytomegalovirus Immunoevasion.

Control of human cytomegalovirus (HCMV) depends on CD8+ T cell responses that are shaped by an individual's repertoire of MHC molecules. MHC class I presentation is modulated by a set of HCMV-encoded proteins. Here we show that HCMV immunoevasins differentially impair T cell recognition of epitopes from the same viral antigen, immediate-early 1 (IE-1), that are presented by different MHC class I allotypes. In the presence of immunoevasins, HLA-A- and HLA-B-restricted T cell clones were ineffective, but HLA-C*0702-restricted T cell clones recognized and killed infected cells. Resistance of HLA-C*0702 to viral immunoevasins US2 and US11 was mediated by the alpha3 domain and C-terminal region of the HLA heavy chain. In healthy donors, HLA-C*0702-restricted T cells dominated the T cell response to IE-1. The same HLA-C allotype specifically protected infected cells from attack by NK cells that expressed a corresponding HLA-C-specific KIR. Thus, allotype-specific viral immunoevasion allows HCMV to escape control by NK cells and HLA-A- and HLA-B-restricted T cells, while the virus becomes selectively vulnerable to an immunodominant population of HLA-C-restricted T cells. Our work identifies a T cell population that may be of particular efficiency in HCMV-specific immunotherapy

Flow allocation in meshed AC-DC electricity grids

In power systems, flow allocation (FA) methods enable to allocate the usage and costs of the transmission grid to each single market participant. Based on predefined assumptions, the power flow is split into isolated generator-specific or producer-specific sub-flows. Two prominent FA methods, Marginal Participation (MP) and Equivalent Bilateral Exchanges (EBEs), build upon the linearized power flow and thus on the Power Transfer Distribution Factors (PTDFs). Despite their intuitive and computationally efficient concepts, they are restricted to networks with passive transmission elements only. As soon as a significant number of controllable transmission elements, such as high-voltage direct current (HVDC) lines, operate in the system, they lose their applicability. This work reformulates the two methods in terms of Virtual Injection Patterns (VIPs), which allows one to efficiently introduce a shift parameter q to tune contributions of net sources and net sinks in the network. In this work, major properties and differences in the methods are pointed out, and it is shown how the MP and EBE algorithms can be applied to generic meshed AC-DC electricity grids: by introducing a pseudo-impedance ω¯ , which reflects the operational state of controllable elements and allows one to extend the PTDF matrix under the assumption of knowing the current flow in the system. Basic properties from graph theory are used to solve for the pseudo-impedance in dependence of the position within the network. This directly enables, e.g., HVDC lines to be considered in the MP and EBE algorithms. The extended methods are applied to a low-carbon European network model (PyPSA-EUR) with a spatial resolution of 181 nodes and an 18% transmission expansion compared to today’s total transmission capacity volume. The allocations of MP and EBE show that countries with high wind potentials profit most from the transmission grid expansion. Based on the average usage of transmission system expansion, a method of distributing operational and capital expenditures is proposed. In addition, it is shown how injections from renewable resources strongly drive country-to-country allocations and thus cross-border electricity flows

Characterization and clinical enrichment of HLA-C*07:02-restricted Cytomegalovirus-specific CD8+ T cells.

Human Cytomegalovirus (CMV) reactivation remains a major source of morbidity in patients after solid organ and hematopoietic stem cell transplantation (HSCT). Adoptive T cell therapy (ACT) with CMV-specific T cells is a promising therapeutic approach for HSCT recipients, but might be counteracted by CMV's immune evasion strategies. HLA-C*07:02 is less susceptible to viral immune evasion suggesting HLA-C*07:02-restricted viral epitopes as promising targets for ACT. For a better understanding of HLA-C*07:02-restricted CMV-specific T cells we used recently generated reversible HLA-C*07:02/IE-1 multimers (Streptamers) recognizing a CMV-derived Immediate-Early-1 (IE-1) epitope and analyzed phenotypic and functional T cell characteristics. Initially, we detected very high frequencies of HLA-C*07:02/IE-1 multimer+ T cells (median = 11.35%), as well as robust functional responses after stimulation with IE-1 peptide (IFNγ+; median = 5.02%) in healthy individuals. However, MHC-multimer+ and IFNγ-secreting T cell frequencies showed a relatively weak correlation (r2 = 0.77), which could be attributed to an unexpected contribution of CMV-epitope-independent KIR2DL2/3-binding of HLA-C*07:02/IE-1 multimers. Therefore, we developed a MHC-multimer double-staining approach against a cancer epitope-specific HLA-C*07:02 multimer to identify truly HLA-C*07:02/IE-1 epitope-specific T cells. The frequencies of these truly HLA-C*07:02/IE-1 multimer+ T cells were still high (median = 6.86%) and correlated now strongly (r2 = 0.96) with IFNγ-secretion. Interestingly, HLA-C*07:02/IE-1-restricted T cells contain substantial numbers with a central memory T cell phenotype, indicating high expansion potential e.g. for ACT. In line with that, we developed a clinical enrichment protocol avoiding epitope-independent KIR-binding to make HLA-C*07:02/IE-1-restricted T cells available for ACT. Initial depletion of KIR-expressing CD8+ T cells followed by HLA-C*07:02/IE-1 Streptamer positive selection using paramagnetic labeling techniques allowed to enrich successfully HLA-C*07:02/IE-1-restricted T cells. Such specifically enriched populations of functional HLA-C*07:02/IE-1-restricted T cells with significant central memory T cell content could become a potent source for ACT

Primary Cytomegalovirus Infection in Seronegative Kidney Transplant Patients Is Associated with Protracted Cold Ischemic Time of Seropositive Donor Organs.

Human Cytomegalovirus (CMV) can lead to primary infection or reactivation in CMV-seronegative or -seropositive kidney transplant recipients, respectively. Complications comprise severe end-organ diseases and acute or chronic transplant rejection. Risk for CMV manifestation is stratified according to the CMV-IgG-serostatus, with donor+/recipient- (D+/R-) patients carrying the highest risk for CMV-replication. However, risk factors predisposing for primary infection in CMV-seronegative recipients are still not fully elucidated. Therefore, we monitored D+/R- high-risk patients undergoing kidney transplantation in combination with antiviral prophylaxis for the incidence of CMV-viremia for a median follow-up time of 784 days (156-1155 days). In this period, we analyzed the functional CMV-specific T cell response by intracellular cytokine staining and CMV-serology by ELISA. Only four of eight D+/R- patients developed clinically relevant CMV-viremia followed by seroconversion. Viremia triggered expansion of functional CMV-specific T cells correlating with protection against secondary CMV-reactivations. In contrast, all other patients remained permanently aviremic and showed no immunological correlate of infection after discontinuation of antiviral prophylaxis for up to three years. Comparing cold ischemic times (CIT) of viremic (median = 1020 min; 720-1080 min) and aviremic patients (median = 335 min; 120-660 min) revealed significantly (p = 0.0286) protracted CIT in patients with primary CMV-infection. Taken together, primary CMV-infection affects only a subgroup of D+/R- patients correlating with length of CIT. Therefore, patients with extended CIT should be thoroughly monitored for CMV-replication well beyond discontinuation of antiviral prophylaxis. In contrast, patients with short CIT remained permanently uninfected and might benefit from shorter prophylactic treatment

HLA-mediated inhibition of NK cell recognition of HCMV infection.

<p>Experiments were performed with polyclonal NK cells (A, C, E, G) or NK cell clone #29 (B, D, F, G) from donor AJU. (A, B) Analysis of KIR expression by flow cytometry. (C, D) Killing by NK cells of the MHC class I-deficient cell lines K562, Daudi and L721.221, HLA-C*0602 or C*0702-transfected L721.221 cells, uninfected MRC-5 fibroblasts, or MRC-5 infected with CMV-wt at moi = 5, at an effector∶target ratio of 2. Data are shown as mean+SD of four replicates from one representative experiment out of four. (E, F) NK cell-mediated killing of uninfected (n.i.) and HCMV-infected fibroblasts over time after infection. Fibroblasts were or were not pretreated with IFN-γ before infection as indicated. (G) Blockade of NK cell mediated-killing by monoclonal antibodies specific for HLA-ABC or KIR2DL2/3 (both IgG2a) or a matched isotype control. Targets were pretreated with IFN-γ. Blockade of the non-KIR ligand HLA-A2 served as additional negative control. The HLA class I type of MRC-5 fibroblasts is HLA-A*0201, A*2902, B*0702, B*4402, C*0501, C*0702. HLA-C*0702 is the only ligand of KIR2DL3 expressed by MRC-5 cells. Killing was assessed at an effector∶target ratio of 2. Data are shown as mean+SD of triplicate samples from one out of two independent experiments.</p

Immunodominance of CRV/C*0702-specific T cells.

<p>(A) Frequencies of CRV-specific T cells in 15 HLA-C*0702-positive blood donors, tested with the CRV nonameric peptide in ELISPOT assays. Black parts of bars indicate CRV-specific signal, grey parts indicate background (no peptide). (B) Specific T cells in PBMCs of HLA-C*0702 carriers were quantified by fluorescent staining with HLA-C*0702/CRV streptamer or HLA-B*0702/TPR pentamer and anti-CD8 antibody. Donors LT12 and SA03 are HCMV-seropositive, donor ASM is HCMV-seronegative. (C) Distribution of T-cell targets within the IE-1 sequence for 15 HLA-C*0702-positive donors, tested with overlapping peptides covering the entire IE-1 sequence of strain AD169. The 120 peptides were divided into 10 subpools, each comprising 12 successive 15-mer peptides with an overlap of 11 amino acids. The C-terminal amino acid position of each subpool is indicated. (D) Frequencies of CRV/C*0702-specific and VLE/A*0201-specific T cells in HLA-C*0702/A*0201-positive donors (n = 6). (E) Comparison of IE-1-specific T cell frequencies in C*0702-negative (n = 13) vs. C*0702-positive (n = 15) donors. (A, C–E) IFN-γ ELISPOT assays were performed with 200 000 peptide-loaded PBMCs in each well and with 2–4 replicates per condition.</p

Effect of HCMV immunoevasins on epitope-specific T cell cytotoxicity.

<p>MRC-5 fibroblasts (HLA-A*0201, C*0702) were preincubated in medium with (A) or without (B) IFN-γ for 72 hours before infection at moi = 5 with AD169 (CMV-wt) or its derivatives CMV-Δall (ΔUS2/3/6/11), CMV-US11 (ΔUS2/3/6) or CMV-US2 (ΔUS3/6/11). Cytotoxicity was determined at 48 hours post infection in a 3.5-hour calcein release assay using an effector∶target ratio of 4. Fibroblasts that were not infected (n.i.) or peptide-loaded (n.i. +peptide) were negative and positive controls, respectively. Data are shown as mean+SD of three to four replicates.</p

Impact of individual HCMV immunoevasins on the recognition of IE-1 T cell epitopes.

<p>MRC-5 fibroblasts were infected with HCMV strain AD169 (wt), with AD169 derivative viruses that expressed only one of the four immunoevasins US2, US3, US6, or US11 as indicated, not infected (n.i.) or peptide-loaded (+pep), and their recognition by T cell clones specific for the IE-1 epitopes CRV/C*0702 and VLE/A*0201 was analyzed. Before infection, fibroblasts were precultured with IFN-γ for 72 hours, then infected at moi = 5 and cocultivated with T cells at 48 hours post infection (10 000 fibroblasts and 10 000 T cells per well). IFN-γ secretion was measured by ELISA. Data are shown as mean+SD of triplicate samples. Representative data are shown for one of 10 CRV-specific clones and one of 4 VLE-specific clones, assayed in two independent experiments.</p

Time course of pp65- and IE-1-specific CD8+ T cell recognition of HCMV-infected fibroblasts.

<p>pp65 and IE-1 T cell epitopes were analyzed for their HLA-A*0201 or -C*0702-restricted presentation to T cell clones at different time points post infection. MRC-5 fibroblasts (HLA-A*0201, C*0702) were not treated (A) or were treated (B) with IFN-γ for 72 hours prior to infection with HCMV AD169 at an moi of 5 or 10. At the indicated time points post infection, 10 000 fibroblasts were incubated with 10 000 T cells for 16–18 hours before measuring antigen-specific IFN-γ secretion by ELISA assay. Cells that were not infected (n.i.) or were peptide-loaded 48 hours post infection (+pep) served as controls. Data are shown as mean+SD of triplicate samples. One of three independent experiments with clones ALT#21 NLV, F61#38 VLE and AJJ#7 CRV is shown, representing experiments with a total of 3 NLV-, 6 VLE- and 12 CRV-specific T cell clones, from 3 different donors for each specificity.</p