SummaryCytomegalovirus (CMV) is a common herpesvirus infection and stimulates the expansion of very large numbers of CMV-specific T cells that reduce the CD4/CD8 ratio and suppress the number of na€ ıve T cells. CMV infection has been associated with frailty and impaired survival. We investigated the correlates of CMV and the impact of the CMV infection on mortality within a cohort of 511 individuals aged at least 65 years who were followed up for 18 years. The mean age of the participants was 74 years of which 70% were CMV-seropositive. CMV was strongly linked to socioeconomic status, and CMV infection increased the annual mortality rate by 42% (Hazard ratio = 1.42, 95% CI: 1.11-1.76 after adjusting for age, sex and baseline socio-economic and health variables) corresponding to 3.7 years lower life expectancy from age 65. Infection was associated with a near doubling of cardiovascular deaths, whereas there was no increase in mortality from other causes. These results show that CMV infection markedly increases the mortality rate in healthy older individuals due to an excess of vascular deaths. These findings may have significant implications for the study of immune senescence and if confirmed more generally could have important implications for measures to optimize the health of the elderly.

The microenvironment of lymphoid organs can aid healthy immune function through provision of both structural and molecular support. In mice, fibroblastic reticular cells (FRCs) create an essential T-cell support structure within lymph nodes, while human FRCs are largely unstudied. Here, we show that FRCs create a regulatory checkpoint in human peripheral T-cell activation through 4 mechanisms simultaneously utilised. Human tonsil and lymph node–derived FRCs constrained the proliferation of both naïve and pre-activated T cells, skewing their differentiation away from a central memory T-cell phenotype. FRCs acted unilaterally without requiring T-cell feedback, imposing suppression via indoleamine-2,3-dioxygenase, adenosine 2A Receptor, prostaglandin E2, and transforming growth factor beta receptor (TGFβR). Each mechanistic pathway was druggable, and a cocktail of inhibitors, targeting all 4 mechanisms, entirely reversed the suppressive effect of FRCs. T cells were not permanently anergised by FRCs, and studies using chimeric antigen receptor (CAR) T cells showed that immunotherapeutic T cells retained effector functions in the presence of FRCs. Since mice were not suitable as a proof-of-concept model, we instead developed a novel human tissue–based in situ assay. Human T cells stimulated using standard methods within fresh tonsil slices did not proliferate except in the presence of inhibitors described above. Collectively, we define a 4-part molecular mechanism by which FRCs regulate the T-cell response to strongly activating events in secondary lymphoid organs while permitting activated and CAR T cells to utilise effector functions. Our results define 4 feasible strategies, used alone or in combinations, to boost primary T-cell responses to infection or cancer by pharmacologically targeting FRCs.

Recent cross‐sectional studies suggest an important role for transitional B lymphocytes (CD19 + CD24hiCD38hi) in promoting transplant tolerance, and protecting from late antibody‐mediated rejection (ABMR). However, prospective studies are lacking. This study enrolled 73 de novo transplant recipients, and collected serial clinical, immunological and biochemical information over 48 ± 6 months. Cell phenotyping was conducted immediately prior to transplantation, and then on five occasions during the first year posttransplantation. When modeled as a time‐dependent covariate, transitional B cell frequencies (but not total B cells or “regulatory” T cells) were associated with protection from acute rejection (any Banff grade; HR: 0.60; 95% CI: 0.37–0.95; p = 0.03). No association between transitional B cell proportions and either de novo donor‐specific or nondonor‐specific antibody (dnDSA; dnNDSA) formation was evident, although preserved transitional B cell proportions were associated with reduced rejection rates in those patients developing dnDSA. Three episodes of ABMR occurred, all in the context of nonadherence, and all associated with in vitro anti‐HLA T cell responses in an ELISPOT assay (p = 0.008 versus antibody‐positive patients not experiencing ABMR). This prospective study supports the potential relevance of transitional (“regulatory”) B cells as a biomarker and therapeutic intervention in transplantation, and highlights relationships between humoral immunity, cellular immunity and nonadherence.

Purpose: Intratumoral hypoxia and immunity have been correlated with patient outcome in various tumor settings. However, these factors are not currently considered for treatment selection in head and neck cancer (HNC) due to lack of validated biomarkers. Here we sought to develop a hypoxiaimmune classifier with potential application in patient prognostication and prediction of response to targeted therapy.Experimental Design: A 54-gene hypoxia-immune signature was constructed on the basis of literature review. Gene expression was analyzed in silico using the The Cancer Genome Atlas (TCGA) HNC dataset (n ¼ 275) and validated using two independent cohorts (n ¼ 130 and 123). IHC was used to investigate the utility of a simplified protein signature. The spatial distribution of hypoxia and immune markers was examined using multiplex immunofluorescence staining.Results: Unsupervised hierarchical clustering of TCGA dataset (development cohort) identified three patient subgroups with distinct hypoxia-immune phenotypes and sur-vival profiles: hypoxia low /immune high , hypoxia high /immune low , and mixed, with 5-year overall survival (OS) rates of 71%, 51%, and 49%, respectively (P ¼ 0.0015). The prognostic relevance of the hypoxia-immune gene signature was replicated in two independent validation cohorts. Only PD-L1 and intratumoral CD3 protein expression were associated with improved OS on multivariate analysis. Hypoxia low / immune high and hypoxia high /immune low tumors were overrepresented in "inflamed" and "immune-desert" microenvironmental profiles, respectively. Multiplex staining demonstrated an inverse correlation between CA-IX expression and prevalence of intratumoral CD3 þ T cells (r ¼ À0.5464; P ¼ 0.0377), further corroborating the transcription-based classification.Conclusions: We developed and validated a hypoxiaimmune prognostic transcriptional classifier, which may have clinical application to guide the use of hypoxia modification and targeted immunotherapies for the treatment of HNC.

Na,K-ATPase is ubiquitously expressed and is essential for maintaining electrochemical and osmotic gradients. The alpha subunit of Na,K-ATPase is the receptor for cardiotonic steroids, which act through the ouabain-binding site and are important in cardiovascular regulation. Interestingly, the presence of endogenous Na,K-ATPase ligands has been implicated in the natriuretic response to perturbations such as hypertension and salt loading; therefore, it is important to characterize the role of the ouabain-binding sites in this context. Because the alpha1 isoform of mice and rats is relatively ouabain resistant, gene-targeting strategies were used to produce mice with reversed responses of the alpha1 and/or alpha2 isoforms to ouabain to assess for altered natriuretic responses to acute salt loading. Regardless of the sensitivity of the alpha2 isoform to ouabain, conferring ouabain sensitivity to alpha1 augmented the natriuretic response to an acute salt load. In addition, when endogenous Na,K-ATPase inhibitors were sequestered with an anti-digoxin antibody fragment, the sodium excretion rates in the ouabain-sensitive alpha1 isoform mice were equivalent to the ouabain-resistant alpha1 isoform mice. These data suggest that the ouabain-binding site of the alpha1 Na,K-ATPase can participate in the natriuretic response to a salt load by responding to endogenous Na,K-ATPase ligands.

Emerging data suggest that expansion of a circulating population of atypical, cytotoxic CD4 + T cells lacking costimulatory CD28 (CD4 + CD28 null cells) is associated with latent cytomegalovirus (CMV) infection. The purpose of the current study was to increase the understanding of the relevance of these cells in 100 unselected kidney transplant recipients followed prospectively for a median of 54 months. Multicolor flow cytometry of peripheral blood mononuclear cells before transplantation and serially posttransplantation was undertaken. CD4 + CD28 null cells were found predominantly in CMV-seropositive patients and expanded in the posttransplantation period. These cells were predominantly effector-memory phenotype and expressed markers of endothelial homing (CX3CR1) and cytotoxicity (NKG2D and perforin). Isolated CD4null cells proliferated in response to peripheral blood mononuclear cells previously exposed to CMV-derived (but not HLA-derived) antigens and following such priming incubation with glomerular endothelium resulted in signs of endothelial damage and apoptosis (release of fractalkine and von Willebrand factor; increased caspase 3 expression). This effect was mitigated by NKG2D-blocking antibody. Increased CD4 + CD28null cell frequencies were associated with delayed graft function and lower estimated glomerular filtration rate at end follow-up. This study suggests an important role for this atypical cytotoxic CD4 + CD28 null cell subset in kidney transplantation and points to strategies that may minimize the impact on clinical outcomes.

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Cytomegalovirus (CMV) infection increases the risk of complications after renal transplantation, but the mechanisms controlling donor-derived infection are not adequately characterized. Here, we assessed the risk of clinically significant CMV disease in donor-seropositive, recipient-seropositive (D+R+) renal transplantation and examined recipients' CMV antigen-specific cellular immune responses primed directly by donor cells. In a retrospective cohort of 569 patients administered standardized basiliximab-tacrolimus-mycophenolate-corticosteroid immunosuppressive therapy, CMV disease rates increased in D+R+ serostatus pairings compared with D2R+ pairings (hazard ratio [HR], 2.61; 95% confidence interval [CI], 1.36 to 5.01; P=0.004) and associated with increased donor-recipient HLA mismatch in the D+R+ group (HR [per class 1 mismatch], 1.43; 95% CI, 1.12 to 1.82]; P=0.02). D+R+ and D+R2 transplants in which the donor and recipient differentially expressed at least one HLA class I allele were followed prospectively from the time of transplantation. During the first year after transplantation, four of eight seropositive recipients and one of three seronegative recipients displayed peripheral blood CD8+ T cell responses to CMV presented by recipient-specific HLA. Notably, no recipients mounted responses to CMV presented by donorspecific HLA, despite the detection of CMV antigen expression in all seropositive donor organs examined (n=10), suggesting that the allograft of Class I HLA-mismatched seropositive donors is inaccessible to CD8+ T cell responses. Finally, pretransplant assays of anti-CMV cellular immunity predicted post-transplant CMV replication less accurately in D+R+ pairings than in D2R+ pairings, possibly reflecting in vitro assay specificity for recipient, rather than donor, HLA. These findings are relevant to the clinical management and immunologic understanding of donortransmitted viral infection.