CD22 is a B cell-specific surface molecule that regulates B cell receptor signaling and serves as a valuable diagnostic and therapeutic target in hematologic malignancies. While CD22 is generally considered restricted to B cells in peripheral blood, reports of off-target staining by certain antibody clones raise concerns regarding clone-dependent cross-reactivity and antibody specificity. Here, we present our comparison of our three anti-CD22 antibody clones to assess their ability to detect intracellular CD22. In the second experiment, we evaluated their specificity for B cells as well as for other blood cell subsets, including dendritic cells and basophils. Our results highlight marked differences among clones in both surface specificity and intracellular labeling performance, underscoring the importance of careful clone selection and validation for accurate CD22 detection.
CD22 (Siglec-2) is a sialic acid-binding immunoglobulin-like lectin predominantly expressed on B cells. As a member of the immunoglobulin superfamily, CD22 functions as a key modulator of B cell receptor (BCR) signaling, primarily exerting an inhibitory effect through recruitment of SHP-1 phosphatase upon ITIM domain phosphorylation. Its expression is generally restricted to mature B cells in peripheral blood and secondary lymphoid tissues, with minimal to absent expression on early progenitors or terminally differentiated plasma cells.
Due to its lineage-specific expression, CD22 has been extensively utilized as a diagnostic marker in flow cytometry panels for B cell identification and as a therapeutic target in B cell malignancies, including acute lymphoblastic leukemia and non-Hodgkin lymphomas. Importantly, CD22 is also the target of several antibody-drug conjugates and CAR-T therapies.
Despite its well-characterized role in B cells, reports of CD22 detection on non-B cell populations, such as dendritic cells or basophils, have emerged, raising questions about potential cross-reactivity of anti-CD22 antibody clones or rare expression under specific conditions. These findings necessitate careful interpretation of flow cytometric data and rigorous validation of antibody specificity.
Here we present our results of experiments aimed at characterizing the specificity of various anti-CD22 antibody clones in human peripheral blood and evaluating potential off-target staining on non-B cell populations such as myeloid dendritic cells (mDCs), plasmacytoid dendritic cells (pDCs), and basophils, as well as the intracellular staining abilities of these clones. At first, we analyzed the staining profile and other staining parameters of four anti-human CD22 clones - MEM-01, IS7, S-HCL-1 and HIB22 on peripheral whole blood of two healthy blood donors. Antibody conjugates with PE were titrated at 3 concentrations (3.75, 1.43 and 0.45 µg/ml). In Fig. 1, Staining profiles at 1.43 µg/ml are shown. In Fig. 2, dot-plot graphs CD22 PE vs. CD19 APC-Cy™7 showing side scatter middle and low leukocytes when stained with all four clones are presented.
Next, we examined specificity of three Exbio CD22 PE conjugates – S-HCL-1, MEM-01 and IS7 together with two CD22 PE conjugate produced with other manufacturer (HIB22, cat. no.: 302506, Biolegend and S-HCL-1, cat. no.: 363503, Biolegend) on human peripheral whole blood of two donors using adaptive immunity and innate immunity panels (based on Kužílková D. et al., Front Immunol. 2022;13:827898). Here we observed three different staining profiles. While MEM-01 stained only B cell subsets, two clones (IS7 and HIB22) also stained myeloid dendritic cells, and S-HCL-1 (of both manufacturers) stained previously mentioned together with plasmacytoid dendritic cells and basophils (see Fig. 3, 4).
These differences may be important to consider, when you are choosing the right clone for your experiment. Broader-specificity anti-CD22 clones (e.g., those that recognize variant or conformationally altered epitopes and may also stain populations like mDCs, pDCs, or basophils) can offer practical advantages in a diagnostic of minimal residual disease setting by increasing sensitivity. They are less likely to miss leukemic or lymphoma B cell populations that have undergone epitope loss, alternative glycosylation, or other surface modifications that would abrogate binding of narrow-specific clones. In heterogeneous or evolving malignant clones, such “pluripotent” recognition can help ensure that aberrant B cells are not falsely categorized as negative due to subtle molecular variation, especially when interpreted within multiparameter panels that discriminate lineage. However, because these clones may also stain non-B cell populations such as mDC, pDC or basophils, their use requires careful multiparametric gating. CD22 should therefore always be interpreted in combination with other B-cell lineage markers, most commonly CD19, or, in the setting of CD19 loss after immunotherapy, with cytoplasmic CD79a and surface CD24, which remain highly stable across B cell development and in leukemic blasts. This strategy preserves the sensitivity benefit of broad CD22 staining while maintaining lineage specificity for reliable MRD assessment.
In contrast, narrow-specificity clones that strictly recognize canonical B-cell CD22 epitopes provide higher precision and lower background, making them preferred for mechanistic studies, lineage confirmation, or when specificity is critical (e.g., distinguishing true CD22 expression from cross-reactive signals on non-B cell populations). They reduce the risk of false positives and simplify interpretation in contexts where off-target staining would confound results. Alternatively, combining both types, using a sensitive, broader-reactive clone for initial detection and a more specific clone for confirmation, alongside orthogonal validation (e.g., transcript analysis or functional assays) - balances sensitivity and specificity.
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