CD28 Function Research Articles

TCF-1 and TOX regulate the memory formation of intestinal group 2 innate lymphoid cells in asthma

Immune memory has been expanded to group 2 innate lymphoid cells (ILC2s), but the cellular and molecular bases remain incompletely understood. Based on house dust mite (HDM)-induced mice asthma models and human samples, we applied flow cytometry, parabiosis, in vivo imaging and adoptive transplantation to confirm the persistence, migration and function of CD45+lineage–CD90.2+NK1.1–NKp46–ST2–KLRG1+IL-17RB+ memory-like ILC2s (ml-ILC2s). Regulated by CCR9/CCL25 and S1P signaling, ml-ILC2s reside in the lamina propria of small intestines (siLP) in asthma remission, and subsequently move to airway upon re-encountering antigens or alarmins. Furthermore, ml-ILC2s possess properties of longevity, potential of rapid proliferation and producing IL-13, and display transcriptional characteristics with up-regulation of Tox and Tcf-7. ml-ILC2s transplantation restore the asthmatic changes abrogated by Tox and Tcf7 knockdown. Our data identify siLP ml-ILC2s as a memory-like subset, which promotes asthma relapse. Targeting TCF-1 and TOX might be promising for preventing asthma recurrence.

An inter-species amino acid variation modulates signaling strength and function of CD28 in vitro and in vivo

Abstract Animal models do not always reflect the human condition. One such example was a phase one clinical trial of a CD28 super agonist antibody (CD28SA). In mice, CD28SA treatment expanded Tregs and reduced autoimmune disease; yet when human volunteers received CD28SA, they experienced life-threatening cytokine storm and multi-organ failure. CD28 is constitutively expressed on T cells and provides the canonical co-stimulatory “signal 2” needed for T cell activation. The proline residues in the CD28 PYAP domain are required for downstream signaling and this motif is conserved across species. However, the human PYAP domain is adjacent to an additional proline, while murine PYAP is followed by an inactive alanine. Previous in vitro studies have suggested this single amino acid variation modulates downstream signaling. We have developed a “humanized” mouse with a CD28 A to P substitution at residue 210 adjacent to the PYAP domain (CD28 A210P). In vitro, CD28 A210PT cells had increased p-Akt, JunB abundance, and produced more IL-2 compared to WT T cells when stimulated in vitro. In vivo, immunization with antigen/CFA emulsion, CD28 A210Pmice display increased antibody production, suggestive of enhanced CD28-mediated Tfh activity. In addition, we assessed the response of the “humanized” CD28 mice to the CD28 super agonist antibody. After injection with CD28 super agonist, CD28 A210Pmice had significantly increased weight loss, elevated and sustained pro-inflammatory cytokine production, and less Treg expansion compared to WT mice. This study suggests interspecies variation in CD28 signaling due to differences outside of the well-studied signaling domains can alter outcomes of CD28 ligation and promote divergent outcomes when targeted in vivo.

Abstract 342: Differential CD45 isoform expression and function in AML versus healthy leukocytes

Abstract CD45 (the leukocyte common antigen) is a phosphatase present on all malignant and non-malignant leukocytes and hematopoietic progenitors and is the most abundant glycoprotein displayed by these cells. The human CD45 gene is alternatively spliced by reassortment of just three exons, exon 4 (“A”), exon 5 (“B”), and exon 6 (“C"), to create five distinct “R” isoforms (RO (no splice exons), RABC, RAB, RBC, and RB), with inclusion of A, B, or C exons enriching for expression of glycans (carbohydrates). Despite decades of investigation, the expression and phosphatase function of these isoforms on human AML cells remains unknown. To address this question, flow cytometry and western blot analysis was performed on AML primary cells (n=3) and KG1a and U937 cell lines versus healthy monocytes (n=3) utilizing exon-specific CD45-targetting mAbs. We then compared the contribution of CD45 phosphatase activity in KG1a and U937 cells versus healthy monocytes by employing a highly specific CD45 phosphatase inhibitor in cell culture for 24hrs, assessing induction of cell death after exposure to the inhibitor (n=3). Our results confirm reports of others indicating that "CD45RA- positivity" is a characteristic phenotype of AML cells, yet, distinctly, our data indicate that the most prevalent isoform identified on AML cells as "CD45RA-positive" is, actually, CD45RABC, the largest and most glycosylated isoform of CD45. This finding is in striking contrast to healthy monocytes, which do not express CD45RABC. Additionally, culturing of human AML cells with a CD45 phosphatase inhibitor induces rapid cell death within 24 hours but does not affect healthy monocytes. Thus, the conspicuous expression of the CD45 isoform CD45RABC among AML cells is associated with a survival advantage. Current studies in AML are directed to elaborating the prevalence of CD45RABC expression and uncovering the downstream effectors of its enzymatic activity to elucidate how such pathways sustain AML growth/survival. Citation Format: Evan Ales, Robert Sackstein. Differential CD45 isoform expression and function in AML versus healthy leukocytes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 342.

Principle and Progress of Treating Lymphoma with CD22 as Immunotherapy Target

B-cell lymphoma is a type of lymphoid tissue tumour, which is usually caused by clonal proliferation of lymphocytes at different stages of differentiation. CD22 molecule is ubiquitous in normal B cells and B cell malignant tumours. It is a principal element in the B cell maturation and proliferation, and provides an important functional link to the adjustment of B cell surface and intracellular signaling and cell survival. Although the function of CD22 remains unclear, blocking the action of CD22 has been proved to inhibit the growth of malignant B cells and CD22 has become an important therapeutic target of B-cell lymphoma. We will also discuss the strategies for the treatment of B-cell lymphoma targeting CD22, including naked antibodies, bispecific antibodies, antibody-drug conjugate (ADC), antibodies conjugated to radioactive elements, and chimeric antigen receptor-based T-cell (CAR-T) cell immunotherapy.

Phenotypic and functional alteration of CD45+ immune cells in the decidua of preeclampsia patients analyzed by mass cytometry (CyTOF).

Preeclampsia (PE) is a severe placenta-related pregnancy disease that has been associated with maternal systemic inflammation and immune system disorders. However, the distribution and functional changes in immune cells of the maternal-placental interface have not been well characterized. Herein, cytometry by time-of-flight mass spectrometry (CyTOF) was used to investigate the immune atlas at the decidua, which was obtained from four PE patients and four healthy controls. Six superclusters were identified, namely, T cells, B cells, natural killer (NK) cells, monocytes, granulocytes, and others. B cells were significantly decreased in the PE group, among which the reduction in CD27+CD38+ regulatory B cell (Breg)-like cells may stimulate immune activation in PE. The significantly increased migration of B cells could be linked to the significantly overexpressed chemokine C-X-C receptor 5 (CXCR5) in the PE group, which may result in the production of excessive autoantibodies and the pathogenesis of PE. A subset of T cells, CD11c+CD8+ T cells, was significantly decreased in PE and might lead to sustained immune activation in PE patients. NK cells were ultimately separated into four subsets. The significant reduction in a novel subset of NK cells (CD56-CD49a-CD38+) in PE might have led to the failure to suppress inflammation at the maternal-fetal interface during PE progression. Moreover, the expression levels of functional markers were significantly altered in the PE group, which also inferred that shifts in the decidual immune state contributed to the development of PE and might serve as potential treatment targets. This is a worthy attempt to elaborate the differences in the phenotype and function of CD45+ immune cells in the decidua between PE and healthy pregnancies by CyTOF, which contributes to understand the pathogenesis of PE, and the altered cell subsets and markers may inspire the immune modulatory therapy for PE.

Erythroid-transdifferentiated myeloid cells promote portal vein tumor thrombus in hepatocellular carcinoma.

Rationale: Hepatocellular carcinoma (HCC) is primarily characterized by a high incidence of vascular invasion. However, the specific mechanism underlying portal vein tumor thrombus (PVTT) in HCC remains unclear. As a consequence of myeloid cell developmental arrest, CD71+ erythroid progenitor cells (EPCs) and myeloid-derived suppressor cells play important roles in HCC; however, their roles in PVTT remain unclear. Methods: The role of CD71+ EPCs in the HCC tumor microenvironment (TME) was evaluated via morphological, RNA-sequencing, enzyme-linked immunosorbent assay, and flow cytometric analyses. Co-culture techniques were employed to assess the CD45+ EPCs and their vascular compromising effect. Additionally, the PVTT-promoting function of CD45+ EPCs was explored in vivo in a murine model. Results: The CD45+EPCs in HCC tissues exhibited increased myeloid cell features, including morphology, surface markers, transforming growth factor (TGF)-β generation, and gene expression, compared with those in circulation. Hence, a large proportion of CD45+EPCs, particularly those in TMEs, comprise erythroid-transdifferentiated myeloid cells (EDMCs). Additionally, the expression of C-C chemokine receptor type 2 (CCR2) mRNA was upregulated in CD45+EPCs within the TME. Tumor macrophages from HCC tissues induced substantial migration of CD45+EPCs in a dose-dependent manner. Meanwhile, results from immunofluorescence analyses revealed that these two cell types are positively associated in the TME and circulation. That is, EDMCs are chemoattracted by HCC macrophages mainly via CCR2 from CD45+ EPCs in the circulation. Additionally, the expressions of FX, FVII, FGB, C4b, CFB, and CFH were elevated in CD45+EPCs within the TME compared with those in the spleen. The CD45+EPCs from the HCC TME promoted vessel endothelial cell migration and compromised tube formation through TGF-β and FGB, respectively. Additionally, CD45+EPCs from the TME induced HCC cell migration. HCC macrophage-induced CD45+EPCs to exhibit higher levels of FX, FVII, FGB, and TGF-β. Meanwhile, upregulation of CCAAT/enhancer binding protein beta expression induced FGB and TGF-β generation in CD45+EPCs in the TME. WTAP, a major RNA m6A writer, stabilized FX and FVII mRNA and enhanced their nuclear export in CD45+EPCs from the TME. CD45+EPCs from the TME were positively associated with PVTT and poor prognosis. Splenectomy reduced the level of CD45+EPCs in the circulation and TME, as well as the incidence of microvascular invasion. The incidence of microvascular invasion increased following the transfer of HCC tissue CD45+EPCs to splenectomized HCC-bearing mice. Conclusions: The CD45+EPCs enriched in the HCC microenvironment are EDMCs, which are induced by HCC macrophages to migrate from the circulation to the TME. Subsequently, EDMCs promote PVTT by compromising the blood vessel endothelium, aggravating coagulation, and promoting HCC cell migration.

Epitope Editing in Hematopoietic Cells Enables CD45-Directed Immune Therapy

Background: Targeted immunotherapies such as CAR-T cells are limited to several well-known lineage markers for which the on-target off-tumor toxicities are clinically tolerated. CART cells are or have been developed individually for different hematologic malignancies based on the relevant lineage antigens (e.g. CD19 for B cell, BCMA for myeloma, CD7 for T cells, CD33 for AML). By targeting a pan-hematologic antigen, a single "drug" could be used for all hematologic indications, thereby accelerating clinical research. CD45 is expressed on most hematologic malignancies and is, therefore, an attractive target for pan-hematologic cancer immunotherapy. However, targeting CD45 with CART cells is limited by (i) expression of CD45 on hematopoietic stem cells (HSC) and their progeny (ii) T cell (including CART cell) fratricide, and (iii) the central function of CD45 in immune cell signaling. To render CD45-specific immunotherapy a potentially viable pan-hematologic CART option we built upon prior work in which we had deleted the pan-myeloid antigen CD33 in HSC. In the current work, however, we sought to modify the relevant CD45 epitope in both T-cells and HSCs in such a way that it is unrecognized by the anti-CD45 CAR-T cells while maintaining its surface expression and intracellular phosphatase function (Figure 1A). Methods: After mapping the amino acid residues on the extracellular domain of human CD45 that are required for binding by three different anti-CD45 scFv's, we devised a CRISPR base editing (BE) approach to install a single mutation at the identified epitope. This allowed us to generate a CD45 molecule that is "invisible" to CAR-T cells made from these anti-CD45 scFv's, thus rendering them resistant to fratricide or suicide. Controls were CD45WT (non-edited) or CD45KO (cells in which CD45 expression was deleted by Cas9-based indel formation). We tested the efficacy of the various CART against leukemia specimens in vitro and in vivo. We then installed the anti-CD45 resistant mutation in primary human CD34+ HSC and assessed the functional consequences of CD45edited hematopoiesis. Results: T-cells transduced with anti-CD45 CARs (CART-45) show substantially impaired ex vivo expansion compared to CD19-directed CAR-T cells due to fratricide, as expected. However, CART-45 cells in which the targeted epitope was altered through adenine base editing (CART-45edited) were resistant to fratricide, enabling the expansion of CD45-directed CAR-T cells comparable to that of CD45KO or CAR19 control T cells. Unlike CD45KO CAR-T cells, CD45editedcells retained CD45 phosphatase expression which is crucial for CAR-T cell function in xenograft tumor models. In mice engrafted with a CD19+ B-ALL cell line, CD45edited CART-19 function was equivalent to CD45WT CART-19, and superior to CD45KO CART-19 (Mock vs CD45KO, p = <0.001); Mock vs CD45edited, p=0.97. These results indicate that CD45 is needed for the proper function of CART cells. We then tested CART-45 in a patient-derived xenograft (PDX) model of human AML and found that CART-45 expanded in the peripheral blood and effectively eliminated tumor cells with no detectable blasts in peripheral blood or bone marrow 4 weeks post CART-45 injection. To protect the hematopoietic system from CD45-directed on-target/off-tumor toxicity, we base-edited the targeted CD45 epitope in human HSCs, achieving the desired Tyr->Cys conversion in 62% of human HSCs (median, 51%-91% range). CD45edited HSC maintained equivalent colony formation in vitro when compared to CD45WT HSC. Crucially, CD45edited HSC transplanted into immunodeficient mice led to equivalent hematopoietic engraftment and differentiation when compared with CD45WT cells, whereas CD45KO hematopoiesis was lost over time (Figure 1B). Conclusions: Here we developed a novel platform that combines pan-hematologic anti-CD45 CAR-T cells with an engineered hematopoietic stem cell transplant endowed with selective resistance to CD45-specific immunotherapy while maintaining CD45 expression and function. The CD45 phosphatase domain is necessary and sufficient to maintain hematopoiesis and T cell function in humanized mice. This platform creates a cancer-specific antigen in all residual host hematopoietic cells, paving the way to profound myeloablation. This platform could be used to treat most hematopoietic malignancies and perhaps other diseases requiring profound hematopoietic ablation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Tetraspanin CD53 controls Tcell immunity through regulation of CD45RO stability, mobility, and function.

T cells depend on the phosphatase CD45 to initiate Tcell receptor signaling. Although the critical role of CD45 in Tcells is established, the mechanisms controlling function and localization in the membrane are not well understood. Moreover, the regulation of specific CD45 isoforms in Tcell signaling remains unresolved. By using unbiased mass spectrometry, we identify the tetraspanin CD53 as a partner of CD45 and show that CD53 controls CD45 function and Tcell activation. CD53-negative Tcells (Cd53-/-) exhibit substantial proliferation defects, and Cd53-/- mice show impaired tumor rejection and reduced IFNγ-producing Tcells compared with wild-type mice. Investigation into the mechanism reveals that CD53 is required for CD45RO expression and mobility. In addition, CD53 is shown to stabilize CD45 on the membrane and is required for optimal phosphatase activity and subsequent Lck activation. Together, our findings reveal CD53 as a regulator of CD45 activity required for Tcell immunity.

Human-like Response of Pig T Cells to Superagonistic Anti-CD28 Monoclonal Antibodies.

Because of its size, anatomical similarities, and now also accessibility to genetic manipulations, pigs are used as animal models for human diseases and immune system development. However, expression and function of CD28, the most important costimulatory receptor expressed by T cells, so far is poorly understood in this species. Using a newly generated mAb (mAb 3D11) with specificity for pig CD28, we detected CD28 on CD8+ and CD4+ αβ T cells. Among γδ T cells, CD28 expression was restricted to a small CD2+ subpopulation of phenotypically naive cells. Functionally, CD28 ligation with mAb 3D11-costimulated porcine T cells, enhanced proliferation and cytokine secretion in vitro. We used a second, likewise newly generated but superagonistic, anti-CD28 mAb (CD28-SA; mAb 4D12) to test the function of CD28 on porcine T cells in a pilot study in vivo. Injection of the CD28-SA into pigs in vivo showed a very similar dose-response relationship as in humans (i.e., 100 µg/kg body weight [BW]) of CD28-SA induced a cytokine release syndrome that was avoided at a dose of 10 µg/kg BW and below. The data further suggest that low-dose (10 µg/kg BW) CD28-SA infusion was sufficient to increase the proportion of Foxp3+ regulatory T cells among CD4+ T cells in vivo. The pig is thus a suitable animal model for testing novel immunotherapeutics. Moreover, data from our pilot study in pigs further suggest that low-dose CD28-SA infusion might allow for selective expansion of CD4+ regulatory T cells in humans.

CD22 Blockage Restores Age-Related Impairments of Microglia Surveillance Capacity.

Microglia, the innate immune cells of the brain, are essential for maintaining homeostasis by their ramified, highly motile processes and for orchestrating the immune response to pathological stimuli. They are implicated in several neurodegenerative diseases like Alzheimer’s and Parkinson’s disease. One commonality of these diseases is their strong correlation with aging as the highest risk factor and studying age-related alterations in microglia physiology and associated signaling mechanism is indispensable for a better understanding of age-related pathomechanisms. CD22 has been identified as a modifier of microglia phagocytosis in a recent study, but not much is known about the function of CD22 in microglia. Here we show that CD22 surface levels are upregulated in aged versus adult microglia. Furthermore, in the amyloid mouse model PS2APP, Aβ-containing microglia also exhibit increased CD22 signal. To assess the impact of CD22 blockage on microglia morphology and dynamics, we have established a protocol to image microglia process motility in acutely prepared brain slices from CX3CR1-GFP reporter mice. We observed a significant reduction of microglial ramification and surveillance capacity in brain slices from aged versus adult mice. The age-related decrease in surveillance can be restored by antibody-mediated CD22 blockage in aged mice, whereas surveillance in adult mice is not affected by CD22 inhibition. Moreover to complement the results obtained in mice, we show that human iPSC-derived macrophages exhibit an increased phagocytic capacity upon CD22 blockage. Downstream analysis of antibody-mediated CD22 inhibition revealed an influence on BMP and TGFβ associated gene networks. Our results demonstrate CD22 as a broad age-associated modulator of microglia functionality with potential implications for neurodegenerative disorders.

Aberrant activation of the CD45-Wnt signaling axis promotes stemness and therapy resistance in colorectal cancer cells.

Rationale: Chemoradiation (CRT) is commonly used as an adjuvant or neoadjuvant treatment for colorectal cancer (CRC) patients. However, resistant cells manage to survive and propagate after CRT, increasing the risk of recurrence. Thus, better understanding the mechanism of resistant cancer cells is required to achieve better clinical outcomes.Methods: Here, we explored gene expression profiling of CRC patient tumors to identify therapy resistance genes and discovered that protein tyrosine phosphatase receptor type C (PTPRC), which encodes CD45, was increased in remnant tumor tissues after CRT and correlated with metastasis. Through multiple validations using patient tumors and CRC cell lines, we found for the first time the increase of CD45 expression in CRC (EpCAM+) epithelial cells surviving after CRT. Thus, we investigated the biological role and downstream events of CD45 were explored in human CRC cells and CRC mouse models.Results: Increased CD45 expression in cancer cells in pretreated primary tumors accounts for poor regression and recurrence-free survival in CRT-treated patients. High CD45 expression promotes CRC cell survival upon 5-fluorouracil or radiation treatment, while CD45 depletion sensitizes CRC cells to CRT. Intriguingly, CD45 is preferentially expressed in cancer stem-like cells (CSCs), as determined by spheroid culture and the expression of CSC markers, and is required for the distinct functions of CSCs, such as cancer initiation, repopulation, and metastasis. Mechanistically, CD45 phosphatase activity promotes Wnt transcriptional activity by stabilizing the β-catenin protein, which collectively enhances stemness and the therapy-resistant phenotype.Conclusions: Our results highlight a novel function of CD45 as a mediator of CRT resistance and provide a potential therapy strategy for CRC therapy.

N-Linked Glycosylation Regulates CD22 Organization and Function.

The organization and clustering of cell surface proteins plays a critical role in controlling receptor signaling; however, the biophysical mechanisms regulating these parameters are not well understood. Elucidating these mechanisms is highly significant to our understanding of immune function in health and disease, given the importance of B cell receptor (BCR) signaling in directing B cells to produce antibodies for the clearance of pathogens, and the potential deleterious effects of dysregulated BCR signaling, such as in B cell malignancies or autoimmune disease. One of main inhibitory co-receptors on B cells is CD22, a sialic-acid binding protein, which interacts homotypically with other sialylated CD22 molecules, as well as heterotypically with IgM and CD45. Although the importance of CD22 in attenuating BCR signaling is well established, we still do not fully understand what mediates CD22 organization and association to BCRs. CD22 is highly glycosylated, containing 12 N-linked glycosylation sites on its extracellular domain, the function of which remain to be resolved. We were interested in how these glycosylation sites mediate homotypic vs. heterotypic interactions. To this end, we mutated five out of the six N-linked glycosylation residues on CD22 localized closest to the sialic acid binding site. Glycan site N101 was not mutated as this resulted in lack of CD22 expression. We used dual-color super-resolution imaging to investigate the impact of altered glycosylation of CD22 on the nanoscale organization of CD22 and its association with BCR. We show that mutation of these five glycosylation sites increased the clustering tendency of CD22 and resulted in higher density CD22 nanoclusters. Consistent with these findings of altered CD22 organization, we found that mutation of N-glycan sites attenuated CD22 phosphorylation upon BCR stimulation, and consequently, increased BCR signaling. Importantly, we identified that these sites may be ligands for the soluble secreted lectin, galectin-9, and are necessary for galectin-9 mediated inhibition of BCR signaling. Taken together, these findings implicate N-linked glycosylation in the organization and function of CD22, likely through regulating heterotypic interactions between CD22 and its binding partners.

Toxic function of CD28 involving in the TLR/MyD88 signal pathway in the river pufferfish (Takifugu obscurus) after exposed to tributyltin chloride (TBT-Cl)

Cluster of differentiation 28 (CD28) is a co-stimulatory receptor found on the surface of T cells. Takifugu obscurus is a kind of anadromous fish species. In this study, the full-length sequence of To-CD28 was obtained, including a 672-bp open reading frame that encodes a peptide chain of 223 amino acids. The phylogenetic analysis showed that To-CD28 is similar to the CD28 protein in Takifugu rubripes. The total hematocyte count distinctly decreased after TBT-Cl exposure, showing the adverse effects of TBT-Cl invasion and self-adjusting ability upon To-CD28 accumulation. The production of reactive oxygen species increased, demonstrating the oxidation resistance of T. obscurus when exposed to TBT-Cl. The tissue expression patterns indicated To-CD28 is a widely distributed receptor in T. obscurus. Its high expression in the liver and gill suggests that To-CD28 could be potentially functioned in TBT-Cl toxic process. The mRNA levels of To-CD28 and relative genes in the TLR-MyD88 signal pathway were significantly up-regulated under TBT-Cl exposure. The immunohistochemistry also showed that the To-CD28 protein signal was enhanced under TBT-Cl exposure, which proved that the positive protection of To-CD28 for maintaining homeostasis. Our study indicated that To-CD28 might participate in the toxicity mechanism upon TBT-Cl exposure and regulate homeostasis stability of T. obscurus.

CD22-Binding Synthetic Sialosides Regulate B Lymphocyte Proliferation Through CD22 Ligand-Dependent and Independent Pathways, and Enhance Antibody Production in Mice

Sialic acid-binding immunoglobulin-like lectins (Siglecs) are expressed in various immune cells and most of them carry signaling functions. High-affinity synthetic sialoside ligands have been developed for various Siglecs. Therapeutic potentials of the nanoparticles and compounds that contain multiple numbers of these sialosides and other reagents such as toxins and antigens have been demonstrated. However, whether immune responses can be regulated by monomeric sialoside ligands has not yet been known. CD22 (also known as Siglec-2) is an inhibitory molecule preferentially expressed in B lymphocytes (B cells) and is constitutively bound and functionally regulated by α2,6 sialic acids expressed on the same cell (cis-ligands). Here, we developed synthetic sialosides GSC718 and GSC839 that bind to CD22 with high affinity (IC50 ~100 nM), and inhibit ligand binding of CD22. When B cells are activated by B cell antigen receptor (BCR) ligation, both GSC718 and GSC839 downregulate proliferation of B cells, and this regulation requires both CD22 and α2,6 sialic acids. This result suggests that these sialosides regulate BCR ligation-induced B cell activation by reversing endogenous ligand-mediated regulation of CD22. By contrast, GSC718 and GSC839 augment B cell proliferation induced by TLR ligands or CD40 ligation, and this augmentation requires CD22 but not α2,6 sialic acids. Thus, these sialosides appear to enhance B cell activation by directly suppressing the inhibitory function of CD22 independently of endogenous ligand-mediated regulation. Moreover, GSC839 augments B cell proliferation that depends on both BCR ligation and CD40 ligation as is the case for in vivo B cell responses to antigens, and enhanced antibody production to the extent comparable to CpG oligonuleotides or a small amount of alum. Although these known adjuvants induce production of the inflammatory cytokines or accumulation of inflammatory cells, CD22-binding sialosides do not. Thus, synthetic sialosides that bind to CD22 with high-affinity modulate B cell activation through endogenous ligand-dependent and independent pathways, and carry an adjuvant activity without inducing inflammation.

Identification and Function of Fibrocytes in Skeletal Muscle Injury Repair and Muscular Dystrophy.

We identified and characterized the function of CD45+/collagen I+ fibrocytes in acutely injured skeletal muscle of wild-type (WT) and Ccr2-/- mice, and in quadriceps and diaphragm muscles of mdx5cv mice, a mouse model for Duchenne muscular dystrophy. Fibrocytes were not detected in peripheral blood of WT mice after acute muscle injury or mdx5cv mice. Fibrocytes were detected in acutely injured muscles and in mdx5cv quadriceps and diaphragm muscles. These cells expressed F4/80 and CCR2, and they were mostly Ly6Clo They expressed a low level of collagens but a high level of profibrotic growth factors as compared with i.m. fibroblasts. Fibrocyte expression of collagens and profibrotic growth factors was not increased in Ccr2-/- mice as compared with WT controls. Fibrocyte expression of both proinflammatory and profibrotic cytokines was significantly higher in mdx5cv diaphragm than in mdx5cv quadriceps. In cocultures, fibrocytes from the mdx5cv diaphragm stimulated a higher level of fibroblast expression of extracellular matrix genes than did those from the mdx5cv quadriceps. Our findings suggest that i.m. fibrocytes most likely originate from infiltrating monocytes/macrophages and differentiate within injured muscles. They likely contribute to the normal muscle injury repair by producing growth factors. They do not appear to contribute to the persistent muscle fibrosis associated with poor injury repair in Ccr2-/- mice. However, they likely contribute to the persistent inflammation and progressive fibrosis in the mdx5cv diaphragm.

Nanoscale organization and dynamics of the siglec CD22 cooperate with the cytoskeleton in restraining BCR signalling.

Receptor organization and dynamics at the cell membrane are important factors of signal transduction regulation. Using super-resolution microscopy and single-particle tracking, we show how the negative coreceptor CD22 works with the cortical cytoskeleton in restraining BCR signalling. In naïve B cells, we found endogenous CD22 to be highly mobile and organized into nanodomains. The landscape of CD22 and its lateral diffusion were perturbed either in the absence of CD45 or when the CD22 lectin domain was mutated. To understand how a relatively low number of CD22 molecules can keep BCR signalling in check, we generated Brownian dynamic simulations and supported them with ex vivo experiments. This combined approach suggests that the inhibitory function of CD22 is influenced by its nanoscale organization and is ensured by its fast diffusion enabling a "global BCR surveillance" at the plasma membrane.

An extracatalytic function of CD45 in B cells is mediated by CD22

The receptor-like tyrosine phosphatase CD45 regulates antigen receptor signaling by dephosphorylating the C-terminal inhibitory tyrosine of the src family kinases. However, despite its abundance, the function of the large, alternatively spliced extracellular domain of CD45 has remained elusive. We used normally spliced CD45 transgenes either incorporating a phosphatase-inactivating point mutation or lacking the cytoplasmic domain to uncouple the enzymatic and noncatalytic functions of CD45 in lymphocytes. Although these transgenes did not alter T-cell signaling or development irrespective of endogenous CD45 expression, both partially rescued the phenotype of CD45-deficient B cells. We identify a noncatalytic role for CD45 in regulating tonic, but not antigen-mediated, B-cell antigen receptor (BCR) signaling through modulation of the function of the inhibitory coreceptor CD22. This finding has important implications for understanding how naïve B cells maintain tonic BCR signaling while restraining inappropriate antigen-dependent activation to preserve clonal "ignorance."

Total Body Irradiation in the "Hematopoietic" Dose Range Induces Substantial Intestinal Injury in Non-Human Primates.

The non-human primate has been a useful model for studies of human acute radiation syndrome (ARS). However, to date structural changes in various parts of the intestine after total body irradiation (TBI) have not been systematically studied in this model. Here we report on our current study of TBI-induced intestinal structural injury in the non-human primate after doses typically associated with hematopoietic ARS. Twenty-four non-human primates were divided into three groups: sham-irradiated control group; and total body cobalt-60 (60Co) 6.7 Gy gamma-irradiated group; and total body 60Co 7.4 Gy gamma-irradiated group. After animals were euthanized at day 4, 7 and 12 postirradiation, sections of small intestine (duodenum, proximal jejunum, distal jejunum and ileum) were collected and fixed in 10% formalin. The intestinal mucosal surface length, villus height and crypt depths were assessed by computer-assisted image analysis. Plasma citrulline levels were determined using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Total bone marrow cells were counted and hematopoietic stem/progenitor cells in bone marrow were analyzed by flow cytometer. Histopathologically, all segments exhibited conspicuous disappearance of plicae circulares and prominent atrophy of crypts and villi. Intestinal mucosal surface length was significantly decreased in all intestinal segments on day 4, 7 and 12 after irradiation (P < 0.02-P < 0.001). Villus height was significantly reduced in all segments on day 4 and 7 (P = 0.02-0.005), whereas it had recovered by day 12 (P > 0.05). Crypt depth was also significantly reduced in all segments on day 4, 7 and 12 after irradiation (P < 0.04-P < 0.001). Plasma citrulline levels were dramatically reduced after irradiation, consistent with intestinal mucosal injury. Both 6.7 and 7.4 Gy TBI reduced total number of bone marrow cells. And further analysis showed that the number and function of CD45(+)CD34(+) hematopoietic stem/progenitors in bone marrow decreased significantly. In summary, TBI in the hematopoietic ARS dose range induces substantial intestinal injury in all segments of the small bowel. These findings underscore the importance of maintaining the mucosal barrier that separates the gut microbiome from the body's interior after TBI.

CD22 signal abnormalities in the pathogenesis of immune related pancytopenia

To investigate the expression of CD22 and its downstream signal molecule spleen tyrosine kinase (SYK) and their phosphorylation of B lymphocytes in patients with immune related pancytopenia(IRP), and to explore the role of CD22 in pathogenesis of IRP. The expression of CD22, SYK and their phosphorylation, along with the expression of IgG and IgM, which obtained from B lymphocytes in peripheral blood of 46 patients with IRP(22 new diagnosed and 24 remitted patients returned to normal after treatment), 22 healthy controls and 12 chronic lymphocytic leukemia(CLL) patients from February to December 2014 were analyzed by flow cytometry. And the mRNA expression of CD22 in peripheral blood mononuclear cell was determined by real-time quantitative PCR. The ratios of CD22+ cells and phosphorylated CD22(pCD22)+ cells of B lymphocytes in new diagnosed group (60. 03% ± 20. 94% 71. 32% ± 11. 16%) were significantly higher than those in remission group (46. 92% ± 20. 04%, 55. 82% ± 14. 42%), normal control group (46. 86% ± 17. 78%, 53. 28% ± 14. 76%) and CLL group (39. 74% ± 18. 96%, 59. 07% ± 17.09%) (all P <0.05). The ratios of phosphorylated SYK( pSYK) + cells in the four groups had the same trend (all P <0. 05). The ratio of pCD22+ cells/pSYK+ cells in new diagnosed group was significantly lower than that in normal control group and CLL group (27. 39 (5. 06 - 102. 70) vs 55. 95 (15. 25 - 298. 53), 56. 92(5. 60 - 228. 96), both P <0. 05), and pCD22+ cells positively correlated to pSYK+ cells ( r = 0. 341, P < 0. 05). The expression of IgG in new diagnosed group and remission group was significantly higher than that in normal control group, and the expression of IgM in new diagnosed group was significantly higher than that in normal control group and CLL group (all P <0. 05). The expression levels of CD22 mRNA in new diagnosed group was significantly higher than that in remission group, normal control group and CLL group (all P <0. 05). The BCR signal pathway of B lymphocyte in IRP patients is enhanced, and the quantity and function of CD22 are increased, while which are still insufficient to inhibit B cell proliferation, and these may have some relationships with the pathogenesis of IRP. [Key words] Pancytopenia; Antigens, CD22; Immune related pancytopenia; Spleen tyrosine kinase; Phosphorylation

Ectopic expression of a novel CD22 splice-variant regulates survival and proliferation in malignant T cells from cutaneous T cell lymphoma (CTCL) patients.

CD22 is a member of the Sialic acid-binding Ig-like lectin (Siglec) family of lectins described to be exclusively present in B lymphocytes and B cell-derived neoplasms. Here, we describe a novel splice form of CD22 (designated CD22∆N), which lacks the N-terminal domain as demonstrated by exon-specific RT-PCR and differential recognition by anti-CD22 antibodies. Importantly, CD22∆N mRNA is expressed in skin lesions from 39 out of 60 patients with cutaneous T cell lymphoma (CTCL), whereas few patients (6 out of 60) expresses full-length, wild type CD22 (CD22wt). In addition, IHC staining of tumor biopsies confirmed the expression of CD22 in CD4+ T cells. Moreover, four out of four malignant T cell lines express CD22: Two cell lines express CD22∆N (MyLa2059 and PB2B) and two express CD22wt (MAC-1 and MAC-2A). siRNA-mediated silencing of CD22 impairs proliferation and survival of malignant T cells, demonstrating a functional role for both CD22∆N and CD22wt in these cells.In conclusion, we provide the first evidence for an ectopic expression of CD22 and a novel splice variant regulating malignant proliferation and survival in CTCL. Analysis of expression and function of CD22 in cutaneous lymphomas may form the basis for development of novel targeted therapies for our patients.