Quantitative contributions of TNF receptor superfamily members to CD8+ T-cell responses.

T-cell responses to infections and cancers are regulated by co-signalling receptors grouped into the binary categories of co-stimulation or co-inhibition. The co-stimulation TNF receptor superfamily (TNFRSF) members 4-1BB, CD27, GITR and OX40 have similar signalling mechanisms raising the question of whether they have similar impacts on T-cell responses. Here, we screened for the quantitative impact of these TNFRSFs on primary human CD8+ T-cell cytokine production. Although both 4-1BB and CD27 increased production, only 4-1BB was able to prolong the duration over which cytokine was produced, and both had only modest effects on antigen sensitivity. An operational model explained these different phenotypes using shared signalling based on the surface expression of 4-1BB being regulated through signalling feedback. The model predicted and experiments confirmed that CD27 co-stimulation increases 4-1BB expression and subsequent 4-1BB co-stimulation. GITR and OX40 displayed only minor effects on their own but, like 4-1BB, CD27 could enhance GITR expression and subsequent GITR co-stimulation. Thus, different co-stimulation receptors can have different quantitative effects allowing for synergy and fine-tuning of T-cell responses.

Human CD8+ T Cells Exhibit a Shared Antigen Threshold for Different Effector Responses.

T cells recognizing cognate pMHC Ags become activated to elicit a myriad of cellular responses, such as target cell killing and the secretion of different cytokines, that collectively contribute to adaptive immunity. These effector responses have been hypothesized to exhibit different Ag dose and affinity thresholds, suggesting that pathogen-specific information may be encoded within the nature of the Ag. In this study, using systematic experiments in a reductionist system, in which primary human CD8+ T cell blasts are stimulated by recombinant peptides presented on MHC Ag alone, we show that different inflammatory cytokines have comparable Ag dose thresholds across a 25,000-fold variation in affinity. Although costimulation by CD28, CD2, and CD27 increased cytokine production in this system, the Ag threshold remained comparable across different cytokines. When using primary human memory CD8+ T cells responding to autologous APCs, equivalent thresholds were also observed for different cytokines and killing. These findings imply a simple phenotypic model of TCR signaling in which multiple T cell responses share a common rate-limiting threshold and a conceptually simple model of CD8+ T cell Ag recognition, in which Ag dose and affinity do not provide any additional response-specific information.

Cerebral Venous Drainage in Patients With Space-Occupying Middle Cerebral Artery Infarction: Effects on Functional Outcome After Hemicraniectomy.

Background: Cerebral venous drainage might influence brain edema characteristics and functional outcome of patients with severe ischemic stroke. The purpose of the study was to evaluate whether hypoplasia of transverse sinuses or the internal jugular veins is associated with poor functional outcome in patients with space-occupying middle cerebral artery (MCA) infarction who underwent decompressive surgery. Methods: We performed a retrospective analysis of patients with space-occupying MCA infarction treated with decompressive surgery at our university hospital. The transverse sinuses and the internal jugular veins were evaluated on baseline images and categorized as normal, hypoplastic or occluded. We defined composite variables for ipsilateral, contralateral or any abnormal cerebral venous drainage. We assessed the functional outcome at 12 months with the modified Rankin scale (mRS) score and defined poor functional outcome as mRS scores 5 and 6. Results: We analyzed 88 patients with available baseline imaging data [mean [SD] patient age 53 (±9) years; median[IQR] time to decompressive surgery 31(22-51) h]. At 12 months 44 patients (50%) had a poor outcome. In univariate analysis neither ipsilateral (OR 1.98;95%CI: 0.75-5.40), nor contralateral (OR 1.56;95%CI: 0.59-4.24) or any (OR 1.6; 95%CI: 0.68-3.79) hypoplasia or occlusion of venous drainage were significantly associated with poor functional outcome. In multivariate analyses, higher patient age (OR 1.07;95%CI 1.01-1.14) and baseline stroke severity (OR 3.42;95%CI 1.31-9.40) were independent predictors of poor functional outcome, but not ipsilateral hypoplasia or occlusion of venous drainage (OR 1.31;95%CI 0.47-3.67). Conclusions: The cerebral venous drainage pattern was not significantly associated with poor functional outcome in our cohort of patients with space-occupying MCA infarction who underwent decompressive surgery.

Comparison of T Cell Activities Mediated by Human TCRs and CARs That Use the Same Recognition Domains.

Adoptive T cell therapies have achieved significant clinical responses, especially in hematopoietic cancers. Two types of receptor systems have been used to redirect the activity of T cells, normal heterodimeric TCRs or synthetic chimeric Ag receptors (CARs). TCRs recognize peptide-HLA complexes whereas CARs typically use an Ab-derived single-chain fragments variable that recognizes cancer-associated cell-surface Ags. Although both receptors mediate diverse effector functions, a quantitative comparison of the sensitivity and signaling capacity of TCRs and CARs has been limited due to their differences in affinities and ligands. In this study we describe their direct comparison by using TCRs that could be formatted either as conventional αß heterodimers, or as single-chain fragments variable constructs linked to CD3ζ and CD28 signaling domains or to CD3ζ alone. Two high-affinity TCRs (KD values of ∼50 and 250 nM) against MART1/HLA-A2 or WT1/HLA-A2 were used, allowing MART1 or WT1 peptide titrations to easily assess the impact of Ag density. Although CARs were expressed at higher surface levels than TCRs, they were 10-100-fold less sensitive, even in the absence of the CD8 coreceptor. Mathematical modeling demonstrated that lower CAR sensitivity could be attributed to less efficient signaling kinetics. Furthermore, reduced cytokine secretion observed at high Ag density for both TCRs and CARs suggested a role for negative regulators in both systems. Interestingly, at high Ag density, CARs also mediated greater maximal release of some cytokines, such as IL-2 and IL-6. These results have implications for the next-generation design of receptors used in adoptive T cell therapies.

No benefits of hypothermia in patients treated with hemicraniectomy for large ischemic stroke.

Background Space-occupying middle cerebral artery brain infarcts are associated with the development of brain edema, which may lead to cerebral herniation and death despite early hemicraniectomy. Aims To evaluate the benefit of therapeutic hypothermia in patients with space-occupying cerebral infarction treated with hemicraniectomy within 48 h of stroke onset. Methods Patients aged 18-60 years with space-occupying cerebral infarction treated with hemicraniectomy within 48 h and hypothermia (33-34°C) were selected from a single university hospital between 2001 and 2010 (n = 53). Patients treated with hemicraniectomy alone served as comparison group (n = 58), originating from three randomized controlled trials evaluating the effects of early decompressive surgery (DECIMAL, DESTINY, HAMLET). Primary outcome was the score on the modified Rankin scale at 12 months dichotomized between modified Rankin scale 0-3 and modified Rankin scale 4-6. Secondary outcome measures were modified Rankin scale score 0-4 and survival. Risk ratios were adjusted with Poisson regression. Results Mean patient age was 48 years. Median time from stroke onset to hemicraniectomy was 23.5 h in both treatment groups. Treatment with hypothermia had no effect on the primary outcome (modified Rankin scale 0-3 versus 4-6 (13/53 (25%) versus 24/58 (41%)); adjusted risk ratio 0.66, 95% confidence interval 0.38-1.13). Fewer patients treated with hypothermia had a modified Rankin scale score of 0-4 (21/53 (40%) versus 42/58 (72%); adjusted risk ratio 0.53, 95% confidence interval 0.37-0.76) and fewer patients survived (26/53 (49%) versus 46/58 (79%); adjusted risk ratio 0.60, 95% confidence interval 0.44-0.82). Conclusions In patients with space-occupying cerebral infarction, treatment with hypothermia had no additional benefit on functional outcome compared with treatment with hemicraniectomy alone.

PD-L1 blockade enhances response of pancreatic ductal adenocarcinoma to radiotherapy.

Pancreatic ductal adenocarcinoma (PDAC) is considered a non-immunogenic tumor, and immune checkpoint inhibitor monotherapy lacks efficacy in this disease. Radiotherapy (RT) can stimulate the immune system. Here, we show that treatment of KPC and Pan02 murine PDAC cells with RT and gemcitabine upregulated PD-L1 expression in a JAK/Stat1-dependent manner. In vitro, PD-L1 inhibition did not alter radio- and chemosensitivity. In vivo, addition of anti-PD-L1 to high (12, 5 × 3, 20 Gy) but not low (6, 5 × 2 Gy) RT doses significantly improved tumor response in KPC and Pan02 allografts. Radiosensitization after PD-L1 blockade was associated with reduced CD11b+Gr1+ myeloid cell infiltration and enhanced CD45+CD8+ T-cell infiltration with concomitant upregulation of T-cell activation markers including CD69, CD44, and FasL, and increased CD8:Treg ratio. Depletion of CD8+ T cells abrogated radiosensitization by anti-PD-L1. Blockade of PD-L1 further augmented the effect of high RT doses (12 Gy) in preventing development of liver metastases. Exploring multiple mathematical models reveals a mechanism able to explain the observed synergy between RT and anti-PD-L1 therapy. Our findings provide a rationale for testing the use of immune checkpoint inhibitors with RT in PDAC.