Judith Leitner

Platelet Function Predicts Myocardial Damage in Patients With Acute Myocardial Infarction

Background—Platelet activation is a hallmark of acute coronary syndromes. Numerous lines of evidence suggest a mechanistic link between von Willebrand factor or platelet hyperfunction and myocardial damage in patients with acute coronary syndromes. Thus, we assessed whether platelet function under high shear rates (collagen adenosine diphosphate closure times [CADP-CTs]) measured with the platelet function analyzer (PFA-100) may be enhanced in patients with myocardial infarction (MI) and whether it may predict the extent of myocardial damage as measured by creatine kinase (CK-MB) or troponin T (TnT) levels. Methods and Results—Patients with acute chest pain or symptoms suggestive of acute coronary syndromes (n=216) were prospectively examined at an emergency department. CADP-CT was significantly shorter in patients with MI, particularly in those with an ST-segment-elevation MI (STEMI) compared with the other patient groups (unstable angina, stable coronary artery disease, or controls). Furthermore, CADP-CT and collagen epinephrine–CT at presentation were independent predictors of myocardial damage as measured by CK-MB or TnT. Patients with MI whose CADP-CT values fell in the first quartile had 3-fold higher CK-MB and TnT levels than those in the fourth quartile. Conclusions—Patients with STEMI have significantly enhanced platelet function when measured under high shear rates. CADP-CT is an independent predictor of the severity of MI, as measured by markers of cardiac necrosis. Measurement of platelet function with the PFA-100 may help in the risk stratification of patients presenting with MI.

B7-H3 is a potent inhibitor of human T-cell activation: No evidence for B7-H3 and TREML2 interaction

B7‐H3 belongs to the B7 superfamily, a group of molecules that costimulate or down‐modulate T‐cell responses. Although it was shown that B7‐H3 could inhibit T‐cell responses, several studies – most of them performed in murine systems – found B7‐H3 to act in a costimulatory manner. In this study, we have specifically addressed a potential functional dualism of human B7‐H3 by assessing the effect of this molecule under varying experimental conditions as well as on different T‐cell subsets. We show that B7‐H3 does not costimulate human T cells. In the presence of strong activating signals, B7‐H3 potently and consistently down‐modulated human T‐cell responses. This inhibitory effect was evident when analysing proliferation and cytokine production and affected naïve as well as pre‐activated T cells. Furthermore, we demonstrate that B7‐H3–T‐cell interaction is characterised by an early suppression of IL‐2 and that T‐cell inhibition can be reverted by exogenous IL‐2. Since the triggering receptor expressed on myeloid cells like transcript 2 (TREML2/TLT‐2) has been recently described as costimulatory receptor of murine B7‐H3 we have extensively analysed interaction of human B7‐H3 with TREML2/TLT‐2. In these experiments we found no evidence for such an interaction. Furthermore, our data do not point to a role for murine TREML2 as a receptor for murine B7‐H3.

The capacity of the TNF family members 4-1BBL, OX40L, CD70, GITRL, CD30L and LIGHT to costimulate human T cells

Activating signals generated by members of the tumour necrosis factor receptor superfamily upon interaction with their cognate ligands play important roles in T‐cell responses. Members of the tumour necrosis factor family namely 4‐1BBL, OX40L, CD70, GITRL, LIGHT and CD30L have been described to function as costimulatory molecules by binding such receptors on T cells. Using our recently described system of T‐cell stimulator cells we have performed the first study where all these molecules have been assessed and compared regarding their capacity to costimulate proliferation and cytokine production of human T cells. 4‐1BBL, which we found to be the most potent molecule in this group, was able to mediate sustained activation and proliferation of human T cells. OX40L and CD70 were also strong inducers of T‐cell proliferation, whereas the costimulatory capacity of human GITRL was significantly lower. Importantly CD30L and LIGHT consistently failed to act costimulatory on human T cells, and we therefore suggest that these molecules might be functionally distinct from the costimulatory members of this family.

TIM-3 Does Not Act as a Receptor for Galectin-9

T cell immunoglobulin and mucin protein 3 (TIM-3) is a type I cell surface protein that was originally identified as a marker for murine T helper type 1 cells. TIM-3 was found to negatively regulate murine T cell responses and galectin-9 was described as a binding partner that mediates T cell inhibitory effects of TIM-3. Moreover, it was reported that like PD-1 the classical exhaustion marker, TIM-3 is up-regulated in exhausted murine and human T cells and TIM-3 blockade was described to restore the function of these T cells. Here we show that the activation of human T cells is not affected by the presence of galectin-9 or antibodies to TIM-3. Furthermore, extensive studies on the interaction of galectin-9 with human and murine TIM-3 did not yield evidence for specific binding between these molecules. Moreover, profound differences were observed when analysing the expression of TIM-3 and PD-1 on T cells of HIV-1-infected individuals: TIM-3 was expressed on fewer cells and also at much lower levels. Furthermore, whereas PD-1 was preferentially expressed on CD45RA−CD8 T cells, the majority of TIM-3-expressing CD8 T cells were CD45RA+. Importantly, we found that TIM-3 antibodies were ineffective in increasing anti-HIV-1 T cell responses in vitro, whereas PD-L antibodies potently reverted the dysfunctional state of exhausted CD8 T cells. Taken together, our results are not in support of an interaction between TIM-3 and galectin-9 and yield no evidence for a functional role of TIM-3 in human T cell activation. Moreover, our data indicate that PD-1, but not TIM-3, is a promising target to ameliorate T cell exhaustion.

Duffy antigen modifies the chemokine response in human endotoxemia

Objective:The Duffy receptor is a promiscuous receptor for chemokines and selectively binds CXC and CC chemokines with high affinity. Preclinical data show that presence of the Duffy receptor on red blood cells may influence plasma levels of proinflammatory cytokines and chemokines and be protective during inflammation. This trial was designed to investigate the influence of the Duffy antigen on human inflammation in vivo. Design:Prospective, analyst-blinded clinical trial. Patients:A total of 32 healthy male volunteers: 16 Duffy-positive white subjects and 16 Duffy-negative subjects of African descent. Measurements and Main Results:All subjects received an intravenous bolus of 2 ng/kg endotoxin (lipopolysaccharide). Cytokines, chemokines, and their receptors were quantified by enzyme immunoassay, reverse transcriptase–polymerase chain reaction, and flow cytometry. Results:Plasma levels of tumor necrosis factor, interleukin-6, interleukin-10, and whole blood growth-related oncogen-α, monocyte chemoattractant protein-1, and interleukin-8 messenger RNA increased similarly in both groups after lipopolysaccharide infusion. Monocyte chemoattractant protein-1 peak plasma levels were roughly two-fold higher in Duffy-positive subjects compared with Duffy-negative subjects (16 ng/mL vs. 7 ng/mL, p < .0001). Similarly, growth-related oncogen-α levels were 2.5-fold higher in Duffy-positive subjects 2 hrs after lipopolysaccharide infusion (210 pg/mL vs. 85 pg/mL; p < .001). Erythrocyte-bound monocyte chemoattractant protein-1, growth-related oncogen-α, and interleukin-8 increased 20- to 50-fold in Duffy-positive subjects (p < .00001 vs. baseline). Conclusion:The Duffy antigen substantially alters chemokine concentrations in blood, but it does not have a protective effect during human endotoxemia.

Identification of PD-1 as a Unique Marker for Failing Immune Reconstitution in HIV-1-Infected Patients on Treatment

PD-1 expression on T cells correlates with T-cell exhaustion and disease progression in HIV-infected patients. Previous studies have shown that combinational antiretroviral therapy induced viral suppression results in immune restoration and reduced PD-1 expression. However, a significant number of patients fail to restore CD4 T cells despite suppression of HIV replication below limit of quantification. In this study, we have analyzed PD-1 expression on CD4 and CD8 T cells in patients with poor immune reconstitution despite successful highly active antiretroviral therapy. We found that T cells of such patients express significantly higher levels of PD-1 than patients who had normal recovery of CD4 cells after treatment. In contrast, failing immune reconstitution was not associated with the expression of activation markers, indicating that PD-1 is a unique marker for failing immune reconstitution despite viral suppression. Furthermore, we show that T cells from patients with poor immune recovery differ from T cells of elderly in respect of their marker profile. PD-1 expression negatively correlated with individual CD4 cell counts, and PD-1 expressing T cells were more prone to programmed death ligand-mediated inhibition of T-cell proliferation, indicating that PD-1-mediated T-cell suppression may have a role in impaired immune reconstitution in HIV patients.

Notch is active in Langerhans cell histiocytosis and confers pathognomonic features on dendritic cells

Langerhans cell histiocytosis (LCH) is an enigmatic disease defined by the accumulation of Langerhans cell-like dendritic cells (DCs). In the present study, we demonstrate that LCH cells exhibit a unique transcription profile that separates them not only from plasmacytoid and myeloid DCs, but also from epidermal Langerhans cells, indicating a distinct DC entity. Molecular analysis revealed that isolated and tissue-bound LCH cells selectively express the Notch ligand Jagged 2 (JAG2) and are the only DCs that express both Notch ligand and its receptor. We further show that JAG2 signaling induces key LCH-cell markers in monocyte-derived DCs, suggesting a functional role of Notch signaling in LCH ontogenesis. JAG2 also induced matrix-metalloproteinases 1 and 12, which are highly expressed in LCH and may account for tissue destruction in LCH lesions. This induction was selective for DCs and was not recapitulated in monocytes. The results of the present study suggest that JAG2-mediated Notch activation confers phenotypic and functional aspects of LCH to DCs; therefore, interference with Notch signaling may be an attractive strategy to combat this disease.

Recombinant human antithrombin inhibits thrombin formation and interleukin 6 release in human endotoxemia

We hypothesized that infusion of recombinant human antithrombin without concomitant heparin would have dose‐dependent anticoagulant properties and potentially decrease endotoxin (lipopolysaccharide [LPS])–induced cytokine production. This was a randomized, double‐blind, placebo‐controlled study in parallel groups enrolling 30 healthy male volunteers. The active treatment groups received infusions of recombinant human antithrombin to increase antithrombin levels to 200% and 500% before infusion of 2 ng/kg endotoxin (LPS). Infusion of antithrombin dose‐dependently decreased coagulation (P<.01 by repeated‐measures ANOVA): peak levels of prothrombin fragment (1.8 nmol/L [95% confidence interval (CI), 1.3–2.3 nmol/L] in the 500% antithrombin group and 4.4 nmol/L [95% CI, 2.7–6.2 nmol/L] in the placebo group at 4 hours), thrombin antithrombin complexes (12 μg/L [95% CI, 8–16 μg/L] in the 500% antithrombin group and 34 μg/L [95% CI, 20–48 μg/L] in the placebo group at 4 hours), and D‐dimer (0.2 μg/L [95% CI, 0.1–0.2 μg/L] in the 500% antithrombin group and 0.5 μg/L [95% CI, 0.4–0.7 μg/L] in the placebo group). Recombinant human antithrombin decreased peak interleukin‐6 levels by 40% (222 pg/mL [95% CI, 148–295 pg/mL] and 216 pg/mL [95% CI, 112–320 pg/mL] in the 500% and 200% antithrombin groups, respectively, versus 357 pg/mL [95% CI, 241–474 pg/mL] in the placebo group; P<.001 by ANOVA). Finally, infusion of recombinant human antithrombin rapidly and transiently decreased neutrophil counts (by 19% [95% CI, 8%–30%] in the 500% antithrombin group versus 6% [95% CI, 1%–10%] in the placebo group, P = .002 by Kruskal‐Wallis ANOVA) and monocyte counts (by 30% [95% CI, 16%–44%] in the 500% antithrombin group and 18% [95% CI, 9%‐28%] in the 200% antithrombin group versus 8% [95% CI, 5%‐20%] in the placebo group, P = .04) before LPS challenge, indicating that recombinant human antithrombin directly interacts with these leukocyte subsets. In summary, recombinant human antithrombin dose‐dependently inhibited tissue factor‐triggered coagulation. Effects on leukocytes and inhibition of interleukin‐6 release seem to represent specific pharmacodynamic properties of recombinant human antithrombin.

Receptors and ligands implicated in human T cell costimulatory processes

It is well established that full activation of T cells that recognize antigens requires additional signals. These second signals are generated by the interaction of costimulatory ligands expressed on antigen presenting cells with their receptors on T cells. In addition, T cell activation processes are negatively regulated by inhibitory costimulatory pathways. Interaction of members of the B7 and the TNF superfamilies with members of the CD28 and TNF-R-superfamilies plays major roles in costimulatory processes. However, a large number of molecules that do not belong to these families have been reported to be involved in the generation of T cell costimulatory signals. In addition to well-defined costimulatory pathways, where both receptors and ligands are known, there are many T cell surface molecules that have been described to generate a second signal under certain experimental conditions, f.i. when ligated with antibodies. Furthermore there are several ligands that have been shown to positively or negatively modulate T cell activation by interacting with as of yet unknown T cell receptors. Here we give a comprehensive overview of molecules that have been implicated in human T cell activation processes and propose criteria that define genuine T cell costimulatory pathways.

No evidence for dualism in function and receptors: PD-L2/B7-DC is an inhibitory regulator of human T cell activation.

The B7 family member programmed‐death‐1‐ligand 2 (PD‐L2/B7‐DC) is a ligand for programmed‐death‐receptor 1 (PD‐1), a receptor involved in negative regulation of T cell activation. Several independent studies have reported that PD‐L2, however, can also potently costimulate murine T cells via an additional yet unidentified receptor. In this study, we evaluated the contribution of PD‐L2 to the activation of human T cells using a novel system of engineered T cell stimulators that expresses membrane‐bound anti‐CD3 antibodies. Analyzing early activation markers, cytokine production and proliferation, we found PD‐L2 to consistently inhibit T cell activation. PD‐L2 inhibition affected CD4+ and CD8+ T cells and was not abrogated by costimulation via CD28. Blocking PD‐1 reverted the inhibitory effect of PD‐L2, demonstrating involvement of this pathway. In human T cells, we found no evidence for any of the costimulatory effects described for PD‐L2 in murine systems. In line with our functional data that do not point to stimulatory PD‐L2‐ligands, we show that binding of PD‐L2‐immunoglobulin to activated human T cells is abrogated by PD‐1 antibodies. Our results demonstrate that PD‐L2 negatively regulates human T cell activation and thus might be a candidate molecule for immunotherapeutic approaches aimed to attenuate pathological immune responses.