GPA: Glycophorin A; RH: Rhesus. We conclude that specificity against RBC membrane antigens is not associated with the severity of the disease. sequentially obtained from 40 patients (24 women/16 men, mean age: 56 years [range: 21C98]) treated for AIHA in the French National Referral Center for adult immune cytopenias. Twenty-seven patients had primary wAIHA and 13 had secondary. Samples were obtained either recently after diagnosis (n=10), during (n=9), or after treatment (n=33). A full description of the cohort and of the methods are described in cellular investigations. Clinical management of the patients as a function of autoAb specificity. Hb levels of patients at the time of diagnosis (on the left), the nadir of Hb (in the center), and the number of lines of therapy (on the right) are indicated and represented as Boxes/Whiskers (min to max values); the horizontal bar in each box indicates the median. GPA: Glycophorin A; RH: Rhesus. We conclude that PRDI-BF1 specificity against RBC membrane antigens is not associated with the severity of the disease. Phagocytosis of autoAb coated RBCs in wAIHA may be the principal pathogenic mechanism underlying RBC destruction, except under certain conditions in which macrophages simply remove clusters of cell-bound IgG on RBCs coated with Clobetasol autoAbs; this phenomenon is called antigen-shaving or trogocytosis.11,12 The testing of phagocytic reactivity of monocytes with Ab-coated RBCs is a useful parameter for evaluating immune RBC destruction in the diagnosis of AIHA or evaluation of the patient response to treatment.13 For this test, normal adherent monocytes were overlaid onto RBCs, sensitized, or not, with eluted autoAbs and incubated for 2 hours at 37C. Slides were then observed under a light microscope for the assessment of RBC phagocytosis and adhesion. Phagocytosis by monocytes was highly variable among the eluates tested (Figure 2A). The tested antibodies demonstrated a very large spectrum in their capacity to Clobetasol induce phagocytosis by CD14+ monocytes: up to 50C60%, and even higher for seven autoAbs, almost higher than that of the positive controls of the test (Figure 2B). The phagocytic index of five individual eluates (one with specificity to GPA and two to RH) were Clobetasol very low, near or below (n=3) the baseline cutoff (Figure 2C). This variation may be due to varying IgG levels in the eluates as well as varying affinities of IgG for RBCs even though the eluates were assessed at comparable dilutions. We observed a similar range of variation for autoAbs against RH, Band Clobetasol 3, and GPA. The data compiled from 25 eluates showed a trend, although not significant (R2=0.1143; MFI of 56 for T0, Figure 3A). Nevertheless, RBCs pre-coated with autoAbs induced a substantial level of membrane exchange with CD14+ monocytes, which varied among the Clobetasol tested eluates, independently of the specificity of the autoAb (Figure 3B). The results of the CD14+ trogocytosis experiments are represented as a MFI index ratio corresponding to the MFI for Ab-coated-RBCs/MFI for non-sensitized RBCs following incubation. There was no correlation between the results of monocyte-mediated RBC trogocytosis and either the Hb level of the patients (R2=0.051; for the representation of the inhibition index obtained for each specificity). Nevertheless, RBC trogocytosis was not totally abolished by a pan Fc-blocking mixture, with persistent intracellular PKH-67 staining detected in CD14+ monocytes (Figure 3D); we also observed this result using higher concentrations of the pan Fc-Blocker (five-fold tested; em data not shown /em ).4 This suggests that RBC membrane transfer to monocytes might contribute to RBC destruction by a mechanism other than classical Fc-mediated phagocytosis because intracellular RBC components were still observed after Fc-Blocker pretreatment. It has been shown that SIRP also plays a role in the removal of aged RBCs through CD47 binding.14 The exact mechanism explaining this observation remains to be investigated. We also assessed antibody-dependent cell-mediated cytotoxicity (ADCC) of PBMC subsets towards sensitized-RBCs through degranulation using transient CD107a exposure at their cell surface.15 Only very low levels of CD107a staining were detected on CD3?CD16/56+ natural killer (NK) cells for most of the autoAbs tested (n=25). We also assessed ADCC of CD3+CD16/56+ natural killer T (NKT) cells and CD3+/CD8+ T cells, which was negative (data not shown). Most values obtained for NK cells were just above or below the estimated baseline cutoff ( em Online Supplementaly Figure S3A /em ). Two autoAbs induced weak CD107a MFI in NK cells and two others (one specific to GPA, the other to Band 3) triggered modest ADCC activity (above the baseline cutoff).