Cellular Requirements for Renal Allograft Rejection

Graft rejection remains a major problem in clinical renal transplantation despite recent improvements in immunosuppressive therapy. While it is accepted that T lymphocytes play an essential role in acute rejection, the relative contributions of the different effector pathways have not been established. The aim of this thesis was to examine the cellular effector mechanisms of renal allograft rejection in the rat. In initial experiments, the characteristic features of unmodified rejection were observed in a serial immunohistological study of rejecting DA allografts transplanted into PVG recipients. The progressive mononuclear cell infiltration of the grafts initially comprised predominantly CD4 lymphocytes and subsequently CD8 cells. In addition to T cells, many of the infiltrating cells were of a phenotype consistent with NK cells and macrophages. This was associated with a striking increase in the expression, within the graft, of donor MHC class I and II antigens, together with the early disappearance from the graft of class II positive donor interstitial cells. Mononuclear cells harvested from the grafts and spleens of these animals displayed moderate levels of in vitro allospecific cytotoxicity against donor strain ConA blasts as well as high levels of nonspecific cytotoxicity against the NK susceptible Y3 target. Following on from these basic observations, subsequent experiments investigated the ability of CD4 or CD8 T lymphocyte subpopulations (prepared by negative selection) to mediate allogeneic kidney damage in different experimental models. One approach was to examine the ability of adoptively transferred lymphocyte subpopulations to cause renal allograft rejection in rats rendered lymphocyte deficient by a sublethal dose of whole body gamma irradiation. Acutely irradiated (8.5 Gy) Lewis recipients were unable to reject DA renal allografts unless reconstituted with syngeneic lymph node cells (LNC). Whereas transfer of 5x10e6 naive Lewis T lymphocytes rapidly restored graft rejection, similar numbers of either CD4 or CDS lymphocytes were relatively ineffective. Immunohistological examination of day 7 allografts in reconstituted recipients revealed, in all groups, a moderate leukocytic infiltrate of similar phenotypic composition, irrespective of the phenotype of the reconstituting cells, and with broad similarity to the infiltrate observed in unmodified rejection. When harvested infiltrating cells and splenocytes were tested in functional assays, only effector cells from CD4+CD8 T cell reconstituted animals, and not those from animals receiving either separated subpopulation, demonstrated allospecific cytotoxicity. Interestingly, splenocytes from all animals (including unreconstituted rats) showed nonspecific cytotoxic activity against Y3, but graft infiltrating cells from the same groups showed none. Collectively, these experiments suggested that both T cell subpopulations were necessary for optimal graft rejection, and that in this particular strain and model, graft rejection correlated with specific cytotoxic I cell lysis rather than nonspecific cytotoxic activity. The second approach examined the ability of T cell subpopulations to mediate allogeneic tissue damage in the renal graft versus host reaction (GVHR). Renal subcapsular injection of parental CD4 lymphocytes in F1 recipients was sufficient to produce, by day 7, a grossly observable renal GVHR, while CD8 lymphocytes (even if specifically sensitised) were ineffective. CD4 lymphocytes required the essential participation of radiosensitive F1 host, bone marrow derived cells to cause tissue damage. This occurred in the absence of demonstrable specific T cell lysis and appeared to be a DTH reaction. Experiments with PVG recombinant rats showed that an isolated MHC class II, but not a class I incompatibility was sufficient to provoke a response. The final group of experiments examined the ability of adoptively transferred lymphocyte subpopulations to restore renal allograft rejection in the congenitally athymic PVG-rnu/rnu rat. CD4 lymphocytes alone were able to restore the first-set rejection response, while CD8 cells alone (naive or specifically sensitised) were ineffective, although the addition of CD8 cells to the inoculum had a synergistic effect on the ability of CD4 cells to restore rejection. Immunohistological studies revealed moderate cellular infiltration of non-rejecting grafts and increased cellular infiltration of the rejecting grafts, with a significant increase in the number of MRC 0X8 positive cells. An interesting finding was the presence, in unmodified PVG-rnu/rnu rats, of extrathymically derived cells with wide alloreactivity as detected by in vitro cytotoxicity assays against a range of allogeneic ConA blasts and NK susceptible targets. Treatment of the effector cells with anti-asialo GM1, and cold target inhibition assays together suggested the presence of populations of atypical, widely reactive NK cells, with the additional ability to preferentially recognise a specific target. These cells were also present in the rejecting grafts of CD4 reconstituted recipients. Overall therefore, the transfer of CD8 lymphocytes alone was insufficient to cause tissue damage in allogeneic kidneys in any of the experimental models studied. In contrast, CD4 lymphocytes alone were able to cause extensive parenchymal damage in the renal GVHR, and were sufficient to initiate allograft rejection in athymic recipients, but required the additional presence of CD8 cells to restore rejection in acutely irradiated animals

Augmentation of Recipient Adaptive Alloimmunity by Donor Passenger Lymphocytes within the Transplant.

Chronic rejection of solid organ allografts remains the major cause of transplant failure. Donor-derived tissue-resident lymphocytes are transferred to the recipient during transplantation, but their impact on alloimmunity is unknown. Using mouse cardiac transplant models, we show that graft-versus-host recognition by passenger donor CD4 T cells markedly augments recipient cellular and humoral alloimmunity, resulting in more severe allograft vasculopathy and early graft failure. This augmentation is enhanced when donors were pre-sensitized to the recipient, is dependent upon avoidance of host NK cell recognition, and is partly due to provision of cognate help for allo-specific B cells from donor CD4 T cells recognizing B cell MHC class II in a peptide-degenerate manner. Passenger donor lymphocytes may therefore influence recipient alloimmune responses and represent a therapeutic target in solid organ transplantation.This work was supported by a British Heart Foundation project grant, the National Institute of Health Research Cambridge Biomedical Research Centre and the National Institute of Health Research Blood and Transplant Research Unit. IGH and JMA were supported by a Wellcome Trust Clinical Research Training Fellowship and Raymond and Beverly Sackler Scholarship. KSP was supported by an Academy of Medical Sciences / Wellcome Trust starter grant.This is the final version of the article. It first appeared from Elsevier via https://doi.org/10.1016/j.celrep.2016.04.00

Subject Benchmark Statement Forensic Science

This document is a QAA Subject Benchmark Statement for Forensic Science that defines what can be expected of a graduate in the subject, in terms of what they might know, do and understand at the end of their studies. Subject Benchmark Statements also describe the nature and characteristics of awards in a particular subject or area. Subject Benchmark Statements are published in QAA's capacity as a membership organisation on behalf of the higher education sector. A summary of the Statement is also available on the QAA website

Age-related changes in the ability to switch between temporal and spatial attention

Background: Identifying age-related changes in cognition that contribute towards reduced driving performance is important for the development of interventions to improve older adults’ driving and prolong the time that they can continue to drive. While driving, one is often required to switch from attending to events changing in time, to distribute attention spatially. Although there is extensive research into both spatial attention and temporal attention and how these change with age, the literature on switching between these modalities of attention is limited within any age group. Methods: Age groups (21–30, 40–49, 50–59, 60–69 and 70+ years) were compared on their ability to switch between detecting a target in a rapid serial visual presentation (RSVP) stream and detecting a target in a visual search display. To manipulate the cost of switching, the target in the RSVP stream was either the first item in the stream (Target 1st), towards the end of the stream (Target Mid), or absent from the stream (Distractor Only). Visual search response times and accuracy were recorded. Target 1st trials behaved as no-switch trials, as attending to the remaining stream was not necessary. Target Mid and Distractor Only trials behaved as switch trials, as attending to the stream to the end was required. Results: Visual search response times (RTs) were longer on “Target Mid” and “Distractor Only” trials in comparison to “Target 1st” trials, reflecting switch-costs. Larger switch-costs were found in both the 40–49 and 60–69 years group in comparison to the 21–30 years group when switching from the Target Mid condition. Discussion: Findings warrant further exploration as to whether there are age-related changes in the ability to switch between these modalities of attention while driving. If older adults display poor performance when switching between temporal and spatial attention while driving, then the development of an intervention to preserve and improve this ability would be beneficial

Fatal COVID-19 outcomes are associated with an antibody response targeting epitopes shared with endemic coronaviruses

The role of immune responses to previously seen endemic coronavirus epitopes in severe acute respiratory coronavirus 2 (SARS-CoV-2) infection and disease progression has not yet been determined. Here, we show that a key characteristic of fatal coronavirus disease (COVID-19) outcomes is that the immune response to the SARS-CoV-2 spike protein is enriched for antibodies directed against epitopes shared with endemic beta-coronaviruses, and has a lower proportion of antibodies targeting the more protective variable regions of the spike. The magnitude of antibody responses to the SARS-CoV-2 full-length spike protein, its domains and subunits, and the SARS-CoV-2 nucleocapsid also correlated strongly with responses to the endemic beta-coronavirus spike proteins in individuals admitted to intensive care units (ICU) with fatal COVID-19 outcomes, but not in individuals with non-fatal outcomes. This correlation was found to be due to the antibody response directed at the S2 subunit of the SARS-CoV-2 spike protein, which has the highest degree of conservation between the beta-coronavirus spike proteins. Intriguingly, antibody responses to the less cross-reactive SARS-CoV-2 nucleocapsid were not significantly different in individuals who were admitted to ICU with fatal and non-fatal outcomes, suggesting an antibody profile in individuals with fatal outcomes consistent with an original antigenic sin type-response

Phenotypic Characterization of EIF2AK4 Mutation Carriers in a Large Cohort of Patients Diagnosed Clinically With Pulmonary Arterial Hypertension.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a rare disease with an emerging genetic basis. Heterozygous mutations in the gene encoding the bone morphogenetic protein receptor type 2 (BMPR2) are the commonest genetic cause of PAH, whereas biallelic mutations in the eukaryotic translation initiation factor 2 alpha kinase 4 gene (EIF2AK4) are described in pulmonary veno-occlusive disease/pulmonary capillary hemangiomatosis. Here, we determine the frequency of these mutations and define the genotype-phenotype characteristics in a large cohort of patients diagnosed clinically with PAH. METHODS: Whole-genome sequencing was performed on DNA from patients with idiopathic and heritable PAH and with pulmonary veno-occlusive disease/pulmonary capillary hemangiomatosis recruited to the National Institute of Health Research BioResource-Rare Diseases study. Heterozygous variants in BMPR2 and biallelic EIF2AK4 variants with a minor allele frequency of <1:10 000 in control data sets and predicted to be deleterious (by combined annotation-dependent depletion, PolyPhen-2, and sorting intolerant from tolerant predictions) were identified as potentially causal. Phenotype data from the time of diagnosis were also captured. RESULTS: Eight hundred sixty-four patients with idiopathic or heritable PAH and 16 with pulmonary veno-occlusive disease/pulmonary capillary hemangiomatosis were recruited. Mutations in BMPR2 were identified in 130 patients (14.8%). Biallelic mutations in EIF2AK4 were identified in 5 patients with a clinical diagnosis of pulmonary veno-occlusive disease/pulmonary capillary hemangiomatosis. Furthermore, 9 patients with a clinical diagnosis of PAH carried biallelic EIF2AK4 mutations. These patients had a reduced transfer coefficient for carbon monoxide (Kco; 33% [interquartile range, 30%-35%] predicted) and younger age at diagnosis (29 years; interquartile range, 23-38 years) and more interlobular septal thickening and mediastinal lymphadenopathy on computed tomography of the chest compared with patients with PAH without EIF2AK4 mutations. However, radiological assessment alone could not accurately identify biallelic EIF2AK4 mutation carriers. Patients with PAH with biallelic EIF2AK4 mutations had a shorter survival. CONCLUSIONS: Biallelic EIF2AK4 mutations are found in patients classified clinically as having idiopathic and heritable PAH. These patients cannot be identified reliably by computed tomography, but a low Kco and a young age at diagnosis suggests the underlying molecular diagnosis. Genetic testing can identify these misclassified patients, allowing appropriate management and early referral for lung transplantation

Improved imputation of low-frequency and rare variants using the UK10K haplotype reference panel

Imputing genotypes from reference panels created by whole-genome sequencing (WGS) provides a cost-effective strategy for augmenting the single-nucleotide polymorphism (SNP) content of genome-wide arrays. The UK10K Cohorts project has generated a data set of 3,781 whole genomes sequenced at low depth (average 7x), aiming to exhaustively characterize genetic variation down to 0.1% minor allele frequency in the British population. Here we demonstrate the value of this resource for improving imputation accuracy at rare and low-frequency variants in both a UK and an Italian population. We show that large increases in imputation accuracy can be achieved by re-phasing WGS reference panels after initial genotype calling. We also present a method for combining WGS panels to improve variant coverage and downstream imputation accuracy, which we illustrate by integrating 7,562 WGS haplotypes from the UK10K project with 2,184 haplotypes from the 1000 Genomes Project. Finally, we introduce a novel approximation that maintains speed without sacrificing imputation accuracy for rare variants