MS4A4A: a novel cell surface marker for M2 macrophages and plasma cells.

MS4A4A is a member of the membrane-spanning, four domain family, subfamily A (MS4A) that includes CD20 (MS4A1), FcRß (MS4A2) and Htm4 (MS4A3). Like the first three members of this family, transcription of MS4A4A appears to be limited to hematopoietic cells. To evaluate expression of the MS4A4A protein in hematopoietic cell lineages and subsets we generated monoclonal antibodies against extracellular epitopes for use in flow cytometry. In human peripheral blood we found that MS4A4A is expressed at the plasma membrane in monocytes but not in granulocytes or lymphocytes. In vitro differentiation of monocytes demonstrated that MS4A4A is expressed in immature but not activated dendritic cells, and in macrophages generated in the presence of interleukin-4 ('alternatively activated' or M2 macrophages) but not by interferon-γ and lipopolysaccharide ('classically' activated or M1 macrophages). MS4A4A was expressed in the U937 monocytic cell line only after differentiation. In normal bone marrow, MS4A4A was expressed in mature monocytes but was undetected, or detected at only a low level, in myeloid/monocytic precursors, as well as their malignant counterparts in patients with various subtypes of myeloid leukemia. Although MS4A4A was not expressed in healthy B lymphocytes, it was highly expressed in normal plasma cells, CD138+ cells from multiple myeloma patients, and bone marrow B cells from a patient with mantle cell lymphoma. These findings suggest immunotherapeutic potential for MS4A4A antibodies in targeting alternatively activated macrophages such as tumor-associated macrophages, and in the treatment of multiple myeloma and mantle cell lymphoma.

Reduced T-dependent humoral immunity in CD20-deficient mice.

CD20 is a tetraspanning membrane protein expressed on B lymphocytes. CD20 deficiency in both mice and humans has recently been shown to have deleterious effects on Ab responses to T-independent Ags; however, no effect on T-dependent immunity has been reported. In this study, we used a Cd20⁻/⁻ mouse line to evaluate Ab responses to adeno-associated virus and SRBCs. The neutralizing Ab response to adeno-associated virus was significantly reduced by CD20 deficiency; both primary (IgM) and secondary (IgG1 and IgG2b) responses to SRBC were also reduced in Cd20⁻/⁻ mice, and this was associated with a reduction in the number of germinal center B cells. A successful humoral response requires the integration of intracellular signaling networks that critically rely on calcium mobilization. In this article, we confirm that BCR-mediated calcium mobilization is reduced in Cd20⁻/⁻ murine B cells after BCR stimulation in vitro, and further show that the reduction is due to an effect on calcium influx rather than calcium release from intracellular stores. Calcium-dependent upregulation of CD69 was impaired in CD20-deficient B cells, as was upregulation of CD86. Altogether, this study demonstrates a role for CD20 in B cell activation and T-dependent humoral immunity.

Prospective Validation of ThyroSPEC Molecular Testing of Indeterminate Thyroid Nodule Cytology Following Diagnostic Pathway Optimization.

Background: Molecular testing for cytologically indeterminate thyroid nodules (ITNs) is often reported with incomplete data on clinical assessment and ultrasound malignancy risk (USMR) stratification. This study aimed to clinically validate the diagnostic accuracy of a novel molecular test, assess the incremental preoperative malignancy risk of other clinical factors, and measure the impacts of introducing molecular testing at the population level. Methods: Comprehensive clinical data were collected prospectively for the first 615 consecutive patients with ITNs in a centralized health care system following implementation of a reflexive molecular test. Clinical data include patient history, method of nodule discovery, clinical assessment, USMR, cytology, molecular testing, and surgery or follow-up along with surgeon notes on surgical decision-making. Accuracy of molecular testing and the impact of the introduction of molecular testing were calculated. A multivariable regression model was developed to identify which clinical factors have the most diagnostic significance for ITNs. Results: A locally developed, low-cost molecular test achieved a negative predictive value (NPV) of 76-91% [confidence interval, CI 66-95%] and a positive predictive value (PPV) of 46-65% [CI 37-75%] in ITNs using only residual material from standard liquid cytology fine-needle aspiration (FNA). Sensitivity was highest (80%; [CI 63-92%]) in the American Thyroid Association (ATA) intermediate-suspicion ultrasound category, and lowest (46%; [CI 19-75%]) in the ATA high-suspicion ultrasound category. Following implementation of molecular testing, diagnostic yield increased by 14% (p = 0.2442) and repeat FNAs decreased by 24% (p = 0.05). Mutation was the primary reason for surgery in 76% of resected, mutation-positive patients. High-risk mutations were associated with a 58% (p = 0.0001) shorter wait for surgery. Twenty-six percent of patients with a negative molecular test result underwent surgery. Multivariable regression highlighted molecular testing and USMR as significantly associated with malignancy. Conclusions: Molecular testing improves preoperative risk stratification but requires further stratification for intermediate-risk mutations. Incorporation of clinical factors (especially USMR) with molecular testing may increase the sensitivity for detection of malignancy. Introduction of molecular testing offers some clinical benefits even in a low resection rate setting, and directly influences surgical decision-making. This study illustrates the importance of the local diagnostic pathway in ensuring appropriate integrated use of molecular testing for best outcomes.

Expression of MS4A and TMEM176 Genes in Human B Lymphocytes.

The MS4A gene family in humans includes CD20 and at least 15 other genes. CD20 exists as homo-oligomers in the plasma membrane, however different MS4A proteins expressed in the same cell may hetero-oligomerize. Given the importance of CD20 in B-cell function and as a therapeutic target, we sought to explore the potential for CD20 hetero-oligomerization with other MS4A proteins. We investigated expression in primary human B-cells of the four MS4A genes previously shown to be expressed in human B-cell lines (MS4A4A, MS4A6A, MS4A7, MS4A8B), as well as two genes comprising the closely related TMEM176 gene family, with a view to identifying candidates for future investigation at the protein level. TMEM176A and TMEM176B transcripts were either not detected, or were detected at relatively low levels in a minority of donor B-cell samples. MS4A4A and MS4A8B transcripts were not detected in any normal B-cell sample. MS4A6A and MS4A7 transcripts were detected at low levels in most samples, however the corresponding proteins were not at the plasma membrane when expressed as GFP conjugates in BJAB cells. We also examined expression of these genes in chronic lymphocytic leukemia (CLL), and found that it was similar to normal B-cells with two exceptions. First, whereas MS4A4A expression was undetected in normal B-cells, it was expressed in 1/14 CLL samples. Second, compared to expression levels in normal B-cells, MS4A6A transcripts were elevated in 4/14 CLL samples. In summary, none of the MS4A/TMEM176 genes tested was expressed at high levels in normal or in most CLL B-cells. MS4A6A and MS4A7 were expressed at low levels in most B-cell samples, however the corresponding proteins may not be positioned at the plasma membrane. Altogether, these data suggest that CD20 normally does not form hetero-oligomers with other MS4A proteins and that there are unlikely to be other MS4A proteins in CLL that might provide useful alternate therapeutic targets.

Quantification of CD20 mRNA and protein levels in chronic lymphocytic leukemia suggests a post-transcriptional defect.

Chronic lymphocytic leukemia is less effectively treated than other B cell malignancies with the anti-CD20 agent, rituximab, presumably due, at least in part, to low CD20 expression. CD20 expression is typically measured by flow cytometry, which may not be quantitative. This study was undertaken to measure total CD20 protein in CLL B cells using quantitative immunoblot analysis. The results demonstrated that total CD20 protein levels were consistently decreased by ∼60% in CLL B cells with low CD20 fluorescence staining. Surprisingly, real-time polymerase chain reaction analysis showed that CD20 mRNA levels were normal or close to normal, depending on the comparative B cell population, and did not correlate well with protein expression. We conclude that CD20 protein is substantially decreased in CLL due to a post-transcriptional defect.

Activation-induced endocytosis of the raft-associated transmembrane adaptor protein LAB/NTAL in B lymphocytes: evidence for a role in internalization of the B cell receptor.

Linker for activation of B cell (LAB)/non-T cell activation linker (NTAL) and phosphoprotein associated with glycophospholipid-enriched membrane microdomain (PAG)/Csk-binding protein (Cbp) are raft-associated transmembrane adaptor proteins with distinct functions in immediate/early phases of receptor signaling pathways. Heterogeneous rafts are thought to compartmentalize membrane-associated signaling events. In order to investigate the subcellular localization of LAB/NTAL and PAG/Cbp, they were expressed as fluorescent chimeric fusion proteins in a human B cell line and their distribution was examined, along with the corresponding endogenous proteins, before and after B cell receptor (BCR) stimulation. Both adaptors were distributed predominantly at the plasma membrane in resting cells and co-clustered with other raft-associated proteins; however, they distributed differently in buoyant membranes isolated by either detergent resistance or non-detergent methods, indicating that they might localize to distinct rafts. After activation, LAB/NTAL was internalized and co-localized with the BCR while PAG/Cbp remained on the cell surface. BCR internalization was reduced in LAB/NTAL-deficient murine B cells, suggesting a regulatory role for LAB/NTAL in activation-induced internalization of the BCR. The cytoplasmic domain of LAB/NTAL, and not the transmembrane/juxtamembrane region, was found to be essential for its internalization.

Cholesterol depletion inhibits src family kinase-dependent calcium mobilization and apoptosis induced by rituximab crosslinking.

The monoclonal antibody (mAb) rituximab produces objective clinical responses in patients with B-cell non-Hodgkin's lymphoma and antibody-based autoimmune diseases. Mechanisms mediating B-cell depletion by rituximab are not completely understood and may include direct effects of signalling via the target antigen CD20. Like most but not all CD20 mAbs, rituximab induces a sharp change in the solubility of the CD20 protein in the non-ionic detergent Triton-X-100, reflecting a dramatic increase in the innate affinity of CD20 for membrane raft signalling domains. Apoptosis induced by rituximab hypercrosslinking has been shown to require src family kinases (SFK), which are enriched in rafts. In this report we provide experimental evidence that SFK-dependent apoptotic signals induced by rituximab are raft dependent. Cholesterol depletion prevented the association of hypercrosslinked CD20 with detergent-insoluble rafts, and attenuated both calcium mobilization and apoptosis induced with rituximab. CD20 cocapped with the raft-associated transmembrane adaptor LAB/NTAL after hypercrosslinking with CD20 mAbs, regardless of their ability to induce a change in the affinity of CD20 for rafts. Taken together, the data demonstrate that CD20 hypercrosslinking via rituximab activates SFKs and downstream signalling events by clustering membrane rafts in which antibody-bound CD20 is localized in a high-affinity configuration.