High-resolution quantification of discrete phagocytic events by live cell time-lapse high-content microscopy imaging.

Phagocytosis is a dynamic process central to immunity and tissue homeostasis. Current methods for quantification of phagocytosis largely rely on indirect or static measurements, such as target clearance or dye uptake, and thus provide limited information about engulfment rates or target processing. Improved kinetic measurements of phagocytosis could provide useful, basic insights in many areas. We present a live-cell, time-lapse and high-content microscopy imaging method based on the detection and quantification of fluorescent dye 'voids' within phagocytes that result from target internalization to quantify phagocytic events with high temporal resolution. Using this method, we measure target cell densities and antibody concentrations needed for optimal antibody-dependent cellular phagocytosis. We compare void formation and dye uptake methods for phagocytosis detection, and examine the connection between target cell engulfment and phagolysosomal processing. We demonstrate how this approach can be used to measure distinct forms of phagocytosis, and changes in macrophage morphology during phagocytosis related to both engulfment and target degradation. Our results provide a high-resolution method for quantifying phagocytosis that provides opportunities to better understand the cellular and molecular regulation of this fundamental biological process.

Macrophage hypophagia as a mechanism of innate immune exhaustion in mAb-induced cell clearance.

Macrophage antibody (Ab)-dependent cellular phagocytosis (ADCP) is a major cytotoxic mechanism for both therapeutic unconjugated monoclonal Abs (mAbs) such as rituximab and Ab-induced hemolytic anemia and immune thrombocytopenia. Here, we studied the mechanisms controlling the rate and capacity of macrophages to carry out ADCP in settings of high target/effector cell ratios, such as those seen in patients with circulating tumor burden in leukemic phase disease. Using quantitative live-cell imaging of primary human and mouse macrophages, we found that, upon initial challenge with mAb-opsonized lymphocytes, macrophages underwent a brief burst (<1 hour) of rapid phagocytosis, which was then invariably followed by a sharp reduction in phagocytic activity that could persist for days. This previously unknown refractory period of ADCP, or hypophagia, was observed in all macrophage, mAb, and target cell conditions tested in vitro and was also seen in vivo in Kupffer cells from mice induced to undergo successive rounds of αCD20 mAb-dependent clearance of circulating B cells. Importantly, hypophagia had no effect on Ab-independent phagocytosis and did not alter macrophage viability. In mechanistic studies, we found that the rapid loss of activating Fc receptors from the surface and their subsequent proteolytic degradation were the primary mechanisms responsible for the loss of ADCP activity in hypophagia. These data suggest hypophagia is a critical limiting step in macrophage-mediated clearance of cells via ADCP, and understanding such limitations to innate immune system cytotoxic capacity will aid in the development of mAb regimens that could optimize ADCP and improve patient outcome.

Moderate dose alcohol protects against serum amyloid protein A1-induced endothelial dysfunction via both notch-dependent and notch-independent pathways.

BACKGROUND: Arterial endothelium plays a critical role in maintaining vessel homeostasis and preventing atherosclerotic cardiovascular disease (CVD). Low-to-moderate alcohol (EtOH) consumption is associated with reduced atherosclerosis and stimulates Notch signaling in endothelial cells. The aim of this study was to determine whether EtOH protects the endothelium against serum amyloid A1 (SAA1)-induced activation/injury, and to determine whether this protection is exclusively Notch-dependent. METHODS AND RESULTS: Human coronary artery endothelial cells (HCAEC) were stimulated or not with "pro-atherogenic" SAA1 (1 µM) in the absence or presence of EtOH (25 mM), the Notch ligand DLL4 (3 µg/ml), or the Notch inhibitor DAPT (20 µM). EtOH stimulated Notch signaling in HCAEC, as evidenced by increased expression of the Notch receptor and hrt target genes. Treatment with EtOH alone or stimulation of Notch signaling by DLL4 increased eNOS activity and enhanced HCAEC barrier function as assessed by trans-endothelial electrical resistance. Moreover, EtOH and DLL4 both inhibited SAA1-induced monolayer leakiness, cell adhesion molecule (ICAM, VCAM) expression, and monocyte adhesion. The effects of EtOH were Notch-dependent, as they were blocked with DAPT and by Notch receptor (N1, N4) knockdown. In contrast, EtOH's inhibition of SAA1-induced inflammatory cytokines (IL-6, IFN-γ) and reactive oxygen species (ROS) was Notch-independent, as these effects were unaffected by DAPT or by N1 and/or N4 knockdown. CONCLUSIONS: EtOH at moderate levels protects against SAA1-induced endothelial activation via both Notch-dependent and Notch-independent mechanisms. EtOH's maintenance of endothelium in a nonactivated state would be expected to preserve vessel homeostasis and protect against atherosclerosis development.

Binding of CLL subset 4 B-cell receptor immunoglobulins to viable human memory B lymphocytes requires a distinctive IGKV somatic mutation.

Amino acid replacement mutations in certain CLL stereotyped B-cell receptor (BCR) immunoglobulins (IGs) at defined positions within antigen-binding sites strongly imply antigen selection. Prime examples of this are CLL subset 4 BCR IGs using IGHV4-34/IGHD5-18/IGHJ6 and IGKV2-30/IGKJ2 rearrangements. Conspicuously and unlike most CLL IGs, subset 4 IGs do not bind apoptotic cells. By testing the (auto)antigenic reactivities of subset 4 IGs toward viable lymphoid-lineage cells and specific autoantigens typically bound by IGHV4-34+ IGs, we found IGs from both subset 4 and non-subset 4 IGHV4-34-expressing CLL cases bind naïve B cells. However, only subset 4 IGs react with memory B cells. Furthermore, subset 4 IGs do not bind DNA nor i or I carbohydrate antigens, common targets of IGHV4-34-utilizing antibodies in systemic lupus erythematosus and cold agglutinin disease, respectively. Notably, we found that subset 4 IG binding to memory B lymphocytes depends on an aspartic acid at position 66 of FR3 in the rearranged IGKV2-30 gene; this amino acid residue is acquired by somatic mutation. Our findings illustrate the importance of positive and negative selection criteria for structural elements in CLL IGs and suggest that autoantigens driving normal B cells to become subset 4 CLL cells differ from those driving IGHV4-34+ B cells in other diseases.

Excessive antigen reactivity may underlie the clinical aggressiveness of chronic lymphocytic leukemia stereotyped subset #8.

Subset #8 is a distinctive subset of patients with chronic lymphocytic leukemia (CLL) defined by the expression of stereotyped IGHV4-39/IGKV1(D)-39 B-cell receptors. Subset #8 patients experience aggressive disease and exhibit the highest risk for Richter transformation among all CLL. In order to obtain biological insight into this behavior, we profiled the antigen reactivity and signaling capacity of subset #8 vs other clinically aggressive stereotyped subsets, namely subsets #1 and #2. Twenty-seven monoclonal antibodies (mAbs) from subsets #1, #2, and #8 CLL clones were prepared as recombinant human immunoglobulin G1 and used as primary antibodies in enzyme-linked immunosorbent assays against representatives of the major classes of established antigenic targets for CLL. Subset #8 CLL mAbs exhibited broad polyreactivity as they bound to all antigens tested, in clear contrast with the mAbs from the other subsets. Antigen challenge of primary CLL cells indicated that the promiscuous antigen-binding activity of subset #8 mAbs could lead to significant cell activation, again in contrast to the less responsive CLL cells from subsets #1 and #2. These features constitute a distinctive profile for CLL subset #8, supporting the existence of distinct mechanisms of aggressiveness in different immunogenetic subsets of CLL.

Not all IGHV3-21 chronic lymphocytic leukemias are equal: prognostic considerations.

An unresolved issue in chronic lymphocytic leukemia (CLL) is whether IGHV3-21 gene usage, in general, or the expression of stereotyped B-cell receptor immunoglobulin defining subset #2 (IGHV3-21/IGLV3-21), in particular, determines outcome for IGHV3-21-utilizing cases. We reappraised this issue in 8593 CLL patients of whom 437 (5%) used the IGHV3-21 gene with 254/437 (58%) classified as subset #2. Within subset #2, immunoglobulin heavy variable (IGHV)-mutated cases predominated, whereas non-subset #2/IGHV3-21 was enriched for IGHV-unmutated cases (P = .002). Subset #2 exhibited significantly shorter time-to-first-treatment (TTFT) compared with non-subset #2/IGHV3-21 (22 vs 60 months, P = .001). No such difference was observed between non-subset #2/IGHV3-21 vs the remaining CLL with similar IGHV mutational status. In conclusion, IGHV3-21 CLL should not be axiomatically considered a homogeneous entity with adverse prognosis, given that only subset #2 emerges as uniformly aggressive, contrasting non-subset #2/IGVH3-21 patients whose prognosis depends on IGHV mutational status as the remaining CLL.

TLR-9 and IL-15 Synergy Promotes the In Vitro Clonal Expansion of Chronic Lymphocytic Leukemia B Cells.

Clinical progression of B cell chronic lymphocytic leukemia (B-CLL) reflects the clone's Ag receptor (BCR) and involves stroma-dependent B-CLL growth within lymphoid tissue. Uniformly elevated expression of TLR-9, occasional MYD88 mutations, and BCR specificity for DNA or Ags physically linked to DNA together suggest that TLR-9 signaling is important in driving B-CLL growth in patients. Nevertheless, reports of apoptosis after B-CLL exposure to CpG oligodeoxynucleotide (ODN) raised questions about a central role for TLR-9. Because normal memory B cells proliferate vigorously to ODN+IL-15, a cytokine found in stromal cells of bone marrow, lymph nodes, and spleen, we examined whether this was true for B-CLL cells. Through a CFSE-based assay for quantitatively monitoring in vitro clonal proliferation/survival, we show that IL-15 precludes TLR-9-induced apoptosis and permits significant B-CLL clonal expansion regardless of the clone's BCR mutation status. A robust response to ODN+IL-15 was positively linked to presence of chromosomal anomalies (trisomy-12 or ataxia telangiectasia mutated anomaly + del13q14) and negatively linked to a very high proportion of CD38(+) cells within the blood-derived B-CLL population. Furthermore, a clone's intrinsic potential for in vitro growth correlated directly with doubling time in blood, in the case of B-CLL with Ig H chain V region-unmutated BCR and <30% CD38(+) cells in blood. Finally, in vitro high-proliferator status was statistically linked to diminished patient survival. These findings, together with immunohistochemical evidence of apoptotic cells and IL-15-producing cells proximal to B-CLL pseudofollicles in patient spleens, suggest that collaborative ODN and IL-15 signaling may promote in vivo B-CLL growth.