A C5a-Immunoglobulin complex in chronic lymphocytic leukemia patients is associated with decreased complement activity

Abstract

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in the Western world. The therapeutic approach to CLL includes chemotherapeutic regimens and immunotherapy. Complement-mediated cytotoxicity, which is one of the mechanisms activated by the therapeutic monoclonal antibodies, depends on the availability and activity of the complement (C) system. The aim was to study the structure of circulating C components and evaluate the importance of C5 structural integrity for C activity in CLL patients. Blood samples were collected from 40 naïve CLL patients and 15 normal controls (NC). The Western blot analysis showed abnormal C5 pattern in some CLL patients, while patterns of C3 and C4 were similar in all subjects. Levels of the C activation markers sC5b-9 and C5a were quantified before and after activation via the classical (CP) and alternative (AP) pathways. In patients with abnormal C5, basal levels of sC5b-9 and C5a were increased while activities of the CP and of the CP C5-convertase, the immediate C5-upstream complex, were decreased compared to NC and to patients with normal C5. The data indicate a link between CP activation and apparent C5 alterations in CLL. This provides a potential prognostic tool that may personalize therapy by identifying a sub-group of CLL patients who display an abnormal C5 pattern, high basal levels of sC5b-9 and C5a, and impaired CP activity, and are likely to be less responsive to immunotherapy due to compromised CP activity.

Cell-free IgG-aggregates in plasma of patients with chronic lymphocytic leukemia cause chronic activation of the classical complement pathway

“…The characteristics of the NCgroup, CLL patients and the patients' subgroups, with undetectable or detectable Ig-C5a, are given in Table 1. The levels of C3 were significantly higher (p<0.002) in CLL patients compared to NC, as previously observed [10]. More than 80% of the patients had C3 levels within the normal range (82-185 and 83-193mg/dl for males and females, respectively, Table 1).…”

“…The levels of sC5b-9 were determined in sera before (Basal sC5b-9 levels) and after in-vitro activation induced via the CP or AP, using aggregated IgG or Zymosan, respectively. Activations were performed in DGVB 2+ buffer, as described [10]. DGVB 2+ buffer: 1 mM MgCl 2 , 0.15 mM CaCl 2 , 71 mM NaCl, 0.1% (w/v) gelatin, 2.5% (w/v) dextrose, and 2.47 mM sodium 5 0 ,5 00diethyl barbiturate (pH 7.35).…”

“…For activation, 1 μg from each collected fraction was diluted to 20 μl in column buffer and mixed with 80 μl DGVB 2+ buffer and 100 μl diluted normal serum (5% serum diluted in DGVB 2+ buffer) to give a final concentration of 5 μg/ml for the fraction's proteins and a final serum concentration of 2.5%. Samples were incubated at 37˚C for 45 min (or 2 hours in the experiments with gel-filtration chromatography fractions) and the reaction was stopped as described [10]. In some experiments C1q-depleted serum (Sigma) was used in a concentration of 2.5%, with and without C1q supplementation.…”

“…In these experiments purified C1q (Sigma) was used in a final concentration of 1μg C1q/1 ml serum. Samples were incubated at 37˚C for 2 hours and the reaction was then stopped as described [10]. Zymosan and in-vitro aggregated IgG, which are effective activators of the AP and CP, respectively, were used as controls.…”

“…We have recently demonstrated a subgroup of CLL patients with increased levels of C activation markers and an Ig-C5a complex, and decreased activity of the classical C pathway (CP), suggesting chronic CP activation [10]. The CP is a main activation cascade of the C system, which also includes the alternative (AP) and lectin pathways.…”