Although tumor-specific CD8 T-cell responses often develop in cancer patients, they rarely result in tumor eradication. We aimed at studying directly the functional efficacy of tumor-specific CD8 T cells at the site of immune attack. Tumor lesions in lymphoid and nonlymphoid tissues (metastatic lymph nodes and soft tissue/visceral metastases, respectively) were collected from stage III/IV melanoma patients and investigated for the presence and function of CD8 T cells specific for the tumor differentiation antigen Melan-A/MART-1. Comparative analysis was conducted with peripheral blood T cells. We provide evidence that in vivo-priming selects, within the available naive Melan-A/MART-1-specific CD8 T-cell repertoire, cells with high T-cell receptor avidity that can efficiently kill melanoma cells in vitro. In vivo, primed Melan-A/MART-1-specific CD8 T cells accumulate at high frequency in both lymphoid and nonlymphoid tumor lesions. Unexpectedly, however, whereas primed Melan-A/MART-1-specific CD8 T cells that circulate in the blood display robust inflammatory and cytotoxic functions, those that reside in tumor lesions (particularly in metastatic lymph nodes) are functionally tolerant. We show that both the lymph node and the tumor environments blunt T-cell effector functions and offer a rationale for the failure of tumor-specific responses to effectively counter tumor progression.

Two different types of CpG motif-containing oligonucleotides (CpG ODN) have been described: CpG-A with high induction of IFN- § in plasmacytoid dendritic cells; and CpG-B with little induction of IFN- § , but potent activation of B cells. In this study, we demonstrate that CpG-A fail to activate B cells unless plasmacytoid dendritic cells are present. We identified a new set of CpG ODN sequences which induces high levels of IFN- § in plasmacytoid dendritic cells but remains capable of directly activating B cells. These new CpG ODN (termed CpG-C) are more potent stimulants of B cells than CpG-B due to their ability of directly and indirectly (via plasmacytoid dendritic cells) activating B cells. The sequence of CpG-C combines structural elements of both CpG-A and CpG-B. The most potent sequence, M362, contains a 5'-end 'TCGTCG-motif' and a 'GTCGTT-motif', both of which are present in CpG-B (ODN 2006); a palindromic sequence characteristic for CpG-A (ODN 2216); but no poly G motif required for CpG-A. In conclusion, we defined the first CpG-containing sequences that potently activate both TLR9-expressing immune cell subsets in humans, the plasmacytoid dendritic cell and the B cell. CpG-C may allow for improved therapeutic immuno-modulation in vivo.

To cite this article: Greinacher A, Juhl D, Strobel U, Wessel A, Lubenow N, Selleng K, Eichler P, Warkentin TE. Heparin-induced thrombocytopenia: a prospective study on the incidence, platelet-activating capacity and clinical significance of antiplatelet factor 4/heparin antibodies of the IgG, IgM, and IgA classes. J Thromb Haemost 2007; 5: 1666-73.Summary. Introduction: Platelet-activating antiplatelet factor 4/heparin (anti-PF4/heparin) antibodies are the major cause of heparin-induced thrombocytopenia (HIT). However, the relative utility of functional (platelet activation) vs. antigen [enzymeimmunoassay (EIA)] assays, and the significance of assay discrepancies remain unresolved. Methods: Consecutive patient sera (n = 1650) referred for diagnostic HIT testing were screened prospectively by both the heparin-induced platelet activation (HIPA) test and anti-PF4/heparin EIA -including individual classes (IgG, IgA, IgM) -with clinical correlations studied. Platelet microparticle and annexin-V-binding properties of the sera were also investigated. Results: Only 205 (12.4%) sera tested positive in either the HIPA and/or EIA: 95 (46.3%) were positive in both, 109 (53.1%) were only EIApositive, and, notably, only one serum was HIPA-positive/EIAnegative. Of 185 EIA-positive sera, only 17.6% had detectable IgM and/or IgA without detectable IgG. Among sera positive for EIA IgG, optical density values were higher when the sera were HIPA-positive (1.117 vs. 0.768; P < 0.0001), with widely overlapping values. Two HIPA-positive but EIA-IgG-negative sera became HIPA-negative following IgG depletion, suggesting platelet-activating antibodies against non-PF4-dependent antigens. Clinical correlations showed that HIPA-negative/ EIA-positive patients did not develop thrombosis and had reasons other than HIT to explain thrombocytopenia. IgM/A antibodies did not increase microparticle penetration, but increased annexin-V binding. Conclusions: The anti-PF4/ heparin EIA has high (99%) sensitivity for HIT. However, only about half of EIA-positive patients are likely to have HIT. Anti-PF4/heparin antibodies of IgM/A class and non-PF4-dependent antigens have only a minor role in HIT.

Summary.  Objectives: To assess efficacy and safety of lepirudin in patients with heparin‐induced thrombocytopenia (HIT) in a prospective study (HAT‐3) as well as in a combined analysis of all HAT study data. Patients/methods: Patients with laboratory‐confirmed HIT were treated with lepirudin in three different aPTT‐adjusted dose regimen and during cardiopulmonary bypass (CPB). Endpoints were new thromboembolic complications (TEC), limb amputations, and death and major bleeding. A historical control group (n = 120) was used for comparison. Results: After start of lepirudin in 205 patients treated in HAT‐3, the combined endpoint occurred in 43 (21.0%). Thirty (14.6%) patients died, 10 (4.9%) underwent limb amputation, and 11 (5.4%) new TECs occurred. Major bleeding occurred in 40 patients (19.5%) (seven during CPB surgery). Combining all prospective HAT trials (n = 403), after start of lepirudin treatment, the combined endpoint occurred in 82 patients (20.3%), with 47 deaths (11.7%), 22 limb amputations (5.5%), 30 new TECs (7.4%), and 71 (17.6%) major bleedings. Compared with the historical control group (log‐rank test), the combined endpoint after start of treatment was reduced (29.7% vs. 52.1%, P = 0.0473), primarily because of reduction in new thromboses (11.9% vs. 32.1%, P = 0.0008). Mean lepirudin maintenance doses ranged from 0.07 to 0.11 mg kg−1 h−1. Major bleeding was more frequent in the lepirudin‐treated patients (29.4% vs. 9.1%, P = 0.0148). Conclusions: The rate of new TECs in HIT patients is low after start of lepirudin treatment. The rate of major bleeding of 17.6% might be reduced by reducing the starting dose to 0.1 mg kg−1 h−1.

Heparin can induce heparin-induced thrombocytopenia (HIT). The combined effect of type of surgery (major vs minor) and heparin on this prothrombotic immune reaction to platelet factor 4 (PF4)/ heparin was analyzed. In a randomized, double-blind study, trauma patients receiving low-molecular-weight (LMWH) or unfractionated heparin (UFH) for thrombosis prophylaxis were assessed for PF4/ heparin-antibody seroconversion, HIT, and thrombosis according to type of surgery. The risk for seroconversion was higher than major versus minor surgery odds ratio, 7.98 [95% confidence interval, 2.06-31.00], P ‫؍‬ .003, controlled for potential confounders, as was the risk for HIT (2.2% [95% confidence interval, 0.3%-4.1%] vs 0.0%, P ‫؍‬ .010). During LMWH compared with UFH thromboprophylaxis, HIT (1 of 298 vs 4 of 316; P ‫؍‬ .370) and PF4/heparin seroconversion (1.7% vs 6.6%; P ‫؍‬ .002) were less frequent, driven by differences in patients undergoing major surgery (incidence of HIT: LMWH 0.8% vs UFH 4.0%; P ‫؍‬ .180; seroconversion rates: 4.0% vs 17.0%; P ‫؍‬ .001). After minor surgery, no case of HIT occurred. The severity of trauma and the need for major surgery strongly influence the risk of an anti-PF4/heparin immune response, which is then increased by UFH. In major trauma certoparin may be safer than UFH because it induces HIT-antibody seroconversion, and the corresponding risk of HIT, less frequently. (Blood.

Detection of IgG, IgM and IgA class antibodies by PF4/heparin ELISA yields a positive test result about twice as often as does a platelet activation assay, with only a minority of the additional patients detected likely having HIT. Thus, there is a potential for considerable over-diagnosis of HIT by laboratories that utilize only an ELISA for diagnostic testing.

Background— Lepirudin (Refludan) is a hirudin derivative. It is a direct thrombin inhibitor obtained by recombinant technology from the medicinal leech and is approved for treatment of heparin-induced thrombocytopenia complicated by thrombosis. Because 3 cases of fatal anaphylaxis possibly associated with use of lepirudin have been reported, we initiated an investigation of putative lepirudin-associated anaphylaxis. Methods and Results— Aided by the manufacturer (Schering AG, Berlin, Germany), we used the lepirudin study databases to identify all patients in whom possible anaphylaxis/severe allergy was recorded from 1994 to September 2002. The 26 possible cases identified were reviewed independently by 2 investigators. After excluding patients with mild skin reactions, reactions likely caused by concomitant medications, poorly documented cases, and reactions that did not correspond temporally with lepirudin use, there remained 9 patients judged to have had severe anaphylaxis in close temporal association with lepirudin. All reactions occurred within minutes of intravenous lepirudin administration, with 4 fatal outcomes (3 acute cardiorespiratory arrests, 1 hypotension-induced myocardial infarction). In these 4 cases, a previous uneventful treatment course with lepirudin was identified (1 to 12 weeks earlier). We recorded high-titer IgG-anti-lepirudin antibodies in an additional patient with anaphylaxis. Because lepirudin has been used in ≈35 000 patients, the risk of anaphylaxis is ≈0.015% (5 of 32 500) on first exposure and 0.16% (4 of 2500) in reexposed patients (7.5% estimated reexposures). Conclusion— Lepirudin can cause fatal anaphylaxis, particularly in patients who are treated within 3 months of a previous exposure. The overall risk/benefit assessment of lepirudin as a treatment for heparin-induced thrombocytopenia remains favorable.

This meta-analysis focuses on 2 prospective studies in patients with heparin-induced thrombocytopenia (HIT) and thromboembolic complication (TEC) who were treated with lepirudin (n = 113). Data were compared with those of a historical control group (n = 91). The primary endpoint (combined incidence of death, new TEC, and limb amputation) occurred in 25 lepirudin-treated patients (22.1%; 95% CI, 14.5%-29.8%): 11 died (9.7%; 95% CI, 4.9%-16.8%), 7 underwent limb amputation (6.2%; 95% CI, 2.5%-12.3%), and 12 experienced new TEC (10.6%; 95% CI, 5.8%-18.3%). The risk was highest in the period between diagnosis of HIT and the start of lepirudin therapy (combined event rate per patient day 6.1%). It markedly decreased to 1.3% during lepirudin treatment and to 0.7% in the posttreatment period. From the start of lepirudin therapy to the end of follow-up, lepirudin-treated patients had consistently lower incidences of the combined endpoint than the historical control group (P = .004, log-rank test), primarily because of a reduced risk for new TEC (P = .005). Thrombin–antithrombin levels in the pretreatment period (median, 43.9 μg/L) decreased after the initiation of lepirudin (at 24 hours ± 6 hours; median, 9.18 μg/L.) During treatment with lepirudin, aPTT ratios of 1.5 to 2.5 produced optimal clinical efficacy with a moderate risk for bleeding, aPTT ratios lower than 1.5 were subtherapeutic, and aPTT levels greater than 2.5 were associated with high bleeding risk. Bleeding events requiring transfusion were significantly more frequent in patients taking lepirudin than in historical control patients (P = .02). In conclusion, this meta-analysis provides further evidence that lepirudin is an effective and acceptably safe treatment for patients with HIT.