Complement activation by human red blood cell antibodies: hemolytic potential of antibodies and efficacy of complement inhibitors assessed by a sensitive flow cytometric assay.

BACKGROUND: Therapeutic intervention strategies in complement-mediated hemolytic diseases are still inappropriate, and lethal events cannot be reliably prevented. As an in vitro model of intravascular hemolysis, a sensitive flow cytometric assay was designed using red blood cells (RBCs) of patients with paroxysmal nocturnal hemoglobinuria (PNH) as target cells. Complement activation by human allo- and autoantibodies directed against RBC antigens and the effect of different complement inhibitors were studied. STUDY DESIGN AND METHODS: RBCs of patients with a PNH III RBC clone of more than 20% were coated with different human allo- or autoantibodies. Hemolysis was initiated with pooled normal human AB serum with or without the addition of complement inhibitors. Loss of PNH III RBCs was estimated by flow cytometry. RESULTS: RBC antibodies of 174 different patients representing 37 different specificities were tested for their potency to activate complement. In correlation with blood group specificities roughly three different patterns were observed: 1) strong and regular, 2) sporadic, and 3) weak or absent complement activation. Remarkably strong complement activators were among antibodies directed against high-prevalence blood group antigens. The C5 inhibitor eculizumab abrogated mild but not strong complement activation, even in presence of excess inhibitor. However, this residual complement activity could be further depressed by combining eculizumab with other inhibitors. CONCLUSION: The PNH hemolysis assay offers a sensitive tool for in vitro analyses of classical pathway-mediated complement activation. The recognition of additive effects of complement inhibitors may guide novel intervention strategies against unwanted complement damage.

Monitoring Blood Immune Cells in Patients with Advanced Small Cell Lung Cancer Undergoing a Combined Immune Checkpoint Inhibitor/Chemotherapy.

In this exploratory prospective observational study on 40 small cell lung cancer (SCLC) patients treated with a combination of chemotherapy and immune checkpoint inhibitors, blood immune cells were characterized by multi-color flow cytometry at the baseline and at the third therapy cycle. The numbers of neutrophils and of T-, B-, and NK cells, as well as the frequency of HLA-DRlow monocytes, 6-SulfoLacNAc (slan)+ non-classical monocytes and circulating dendritic cell (DC) subtypes were determined. The prognostic value of the parameters was evaluated by the patient's survival analysis with overall survival (OS) as the primary endpoint. In addition, blood cell parameters from SCLC patients were compared to those from non-SCLC (NSCLC). The global median OS of patients was 10.4 ± 1.1 months. Disease progression (15% of patients) correlated with a higher baseline neutrophil/lymphocyte ratio (NLR), more HLA-DRlow monocytes, and lower NK cell and DC numbers. The risk factors for poor OS were the presence of brain/liver metastases, a baseline NLR ≥ 6.1, HLA-DRlow monocytes ≥ 21% of monocytes, slan+ non-classical monocytes < 0.12%, and/or CD1c+ myeloid DC < 0.05% of leukocytes. Lymphocytic subpopulations did not correlate with OS. When comparing biomarkers in SCLC versus NSCLC, SCLC had a higher frequency of brain/liver metastases, a higher NLR, the lowest DC frequencies, and lower NK cell numbers. Brain/liver metastases had a substantial impact on the survival of SCLC patients. At the baseline, 45% of SCLC patients, but only 24% of NSCLC patients, had between three and five risk factors. A high basal NLR, a high frequency of HLA-DRlow monocytes, and low levels of slan+ non-classical monocytes were associated with poor survival in all lung cancer histotypes. Thus, the blood immune cell signature might contribute to a better prediction of SCLC patient outcomes and may uncover the pathophysiological peculiarities of this tumor entity.

Blood Immune Cell Biomarkers in Lung Cancer Patients Undergoing Treatment with a Combination of Chemotherapy and Immune Checkpoint Blockade.

Although immune checkpoint inhibitor (ICI) therapies have improved the treatment of patients with advanced non-small cell lung cancer (NSCLC), several patients do not achieve durable clinical responses. Biomarkers for the prediction of therapy responses are urgently needed. To identify blood cell parameters correlating with patients' survival, immune cells from 90 patients with NSCLC undergoing a combination of ICI and chemotherapy were prospectively monitored. At the time point of the first and third antibody administration, complete leukocyte blood count, the percentage of HLA-DRlow monocytes, the percentage of 6-Sulfo LacNAc (slan)+CD16+ non-classical monocytes, and the number of circulating dendritic cell (DC) subtypes, as well as T-, B-, and NK cells were determined by multi-color flow cytometry in peripheral blood. The prognostic value of the immune cell parameters investigated was evaluated by patients' survival analysis, with progression-free survival (PFS) as the main criterion. A total of 67 patients (74.4%) showed a partial remission or a stable disease, and 35% of patients even survived 12 months and longer. Patients with a neutrophil-to-lymphocyte ratio (NLR) ≥6.1, a frequency of HLA-DRlow monocytes ≥22%, of slan+ non-classical monocytes <0.25% of leukocytes, and/or a sum of myeloid DC (MDC) and plasmacytoid DC (PDC) ≤0.14% of leukocytes had a poorer prognosis. The hazard ratio for PFS was 2.097 (1.208−3.640) for the NLR, 1.964 (1.046−3.688) for HLA-DRlow monocytes, 3.202 (1.712−5.99) for slan+ non-classical monocytes, and 2.596 (1.478−4.56) for the MDC/PDC sum. Patients without any of the four risk factors showed the best PFS. Furthermore, low NK cell counts correlated with shorter PFS (cutoff 200 cells/µL). Female patients had lower baseline NK cell counts and a shorter PFS. Our study confirms the usefulness of blood immune cells as biomarkers for clinical response and survival in NSCLC patients undergoing a combined ICI/chemotherapy.