CD52/FLAG and CD52/HA Fusion Proteins as Novel Magnetic Cell Selection Markers.

At present, the magnetic selection of genetically modified cells is mainly performed with surface markers naturally expressed by cells such as CD4, LNGFR (low affinity nerve growth factor receptor), and MHC class I molecule H-2Kk. The disadvantage of such markers is the possibility of their undesired and poorly predictable expression by unmodified cells before or after cell manipulation, which makes it essential to develop new surface markers that would not have such a drawback. Earlier, modified CD52 surface protein variants with embedded HA and FLAG epitope tags (CD52/FLAG and CD52/HA) were developed by the group of Dr. Mazurov for the fluorescent cell sorting of CRISPR-modified cells. In the current study, we tested whether these markers can be used for the magnetic selection of transduced cells. For this purpose, appropriate constructs were created in MigR1-based bicistronic retroviral vectors containing EGFP and DsRedExpress2 as fluorescent reporters. Cytometric analysis of the transduced NIH 3T3 cell populations after magnetic selection evaluated the efficiency of isolation and purity of the obtained populations, as well as the change in the median fluorescence intensity (MFI). The results of this study demonstrate that the surface markers CD52/FLAG and CD52/HA can be effectively used for magnetic cell selection, and their efficiencies are comparable to that of the commonly used LNGFR marker. At the same time, the significant advantage of these markers is the absence of HA and FLAG epitope sequences in cellular proteins, which rules out the spurious co-isolation of negative cells.

Anti-CD52 antibody treatment in murine experimental autoimmune encephalomyelitis induces dynamic and differential modulation of innate immune cells in peripheral immune and central nervous systems.

Anti-CD52 antibody (anti-CD52-Ab) leads to a rapid depletion of T and B cells, followed by reconstitution of immune cells with tolerogenic characteristics. However, very little is known about its effect on innate immune cells. In this study, experimental autoimmune encephalomyelitis mice were administered murine anti-CD52-Ab to investigate its effect on dendritic cells and monocytes/macrophages in the periphery lymphoid organs and the central nervous system (CNS). Our data show that blood and splenic innate immune cells exhibited significantly increased expression of MHC-II and costimulatory molecules, which was associated with increased capacity of activating antigen-specific T cells, at first day but not three weeks after five daily treatment with anti-CD52-Ab in comparison with controls. In contrast to the periphery, microglia and infiltrating macrophages in the CNS exhibited reduced expression levels of MHC-II and costimulatory molecules after antibody treatment at both time-points investigated when compared to controls. Furthermore, the transit response of peripheral innate immune cells to anti-CD52-Ab treatment was also observed in the lymphocyte-deficient SCID mice, suggesting the changes are not a direct consequence of the mass depletion of lymphocytes in the periphery. Our study demonstrates a dynamic and tissue-specific modulation of the innate immune cells in their phenotype and function following the antibody treatment. The findings of differential modulation of the microglia and infiltrating macrophages in the CNS in comparison with the innate immune cells in the peripheral organs support the CNS-specific beneficial effect of alemtuzumab treatment on inhibiting neuroinflammation in multiple sclerosis patients.

Mutations in PIGA cause a CD52-/GPI-anchor-deficient phenotype complicating alemtuzumab treatment in T-cell prolymphocytic leukemia.

OBJECTIVE: Infusional alemtuzumab followed by consolidating allogeneic hematopoietic stem cell transplantation in eligible patients is considered a standard of care in T-cell prolymphocytic leukemia (T-PLL). Antibody selection against CD52 has been associated with the development of CD52-negative leukemic T cells at time of relapse. Clinical implications and molecular mechanisms underlying this phenotypic switch are unknown. METHODS: We performed flow cytometry and real-time-PCR for CD52-expression and next generation sequencing for PIGA mutational analyses. RESULTS: We identified loss of CD52 expression after alemtuzumab treatment in two of 21 T-PLL patients resulting from loss of GPI-anchor expression caused by inactivating mutations of the PIGA gene. One patient with relapsed T-PLL exhibited a single PIGA mutation, causing a CD52-negative escape variant of the initial leukemic cell clone, preventing alemtuzumab-retreatment. The second patient with continued complete remission after alemtuzumab treatment harbored three different PIGA mutations that affected either the non-neoplastic T cell or the mononuclear cell compartment and resulted in symptomatic paroxysmal nocturnal hemoglobinuria. Next generation sequencing of T-PLL cells collected before the initiation of treatment revealed PIGA wild-type sequence reads in all 16 patients with samples available for testing. CONCLUSION: These data indicate that PIGA mutations were acquired during or after completion of alemtuzumab treatment.

Anti-mouse CD52 Treatment Ameliorates Colitis through Suppressing Th1/17 Mediated Inflammation and Promoting Tregs Differentiation in IL-10 Deficient Mice.

Recent studies suggested that excessive T helper (Th)1/17 cells concomitant with regulatory T cell deficiency might play important roles in Crohn's disease. Anti-cluster of differentiation 52 (CD52) monoclonal antibody (mAb), which aims on CD52 antigen on mature immunocytes, has both T cell depletion and immunosuppressive activities. In this study, we evaluated the therapeutic effects and possible mechanisms of anti-CD52 treatment on interleukin-10 (IL-10) deficient mouse. Anti-mouse CD52 mAb was administered to C3H/HeJBir.IL-10-/- (C3H.IL-10-/-) mice intraperitoneally 20 µg per week for 2 weeks. The disease activity index, body weight, the histological grading of colitis, and levels of tumor necrosis factor (TNF)-α, interferon (IFN)-γ, IL-17 and IL-6 in colon were quantified after treatment. In addition, CD25, Forkhead box P3 (Foxp3) and transforming growth factor (TGF)-ß gene as well as the percentage of CD25+Foxp3+ T cells in colon were also measured. The severity of colitis in IL-10-/- mice was significantly decreased by the treatment, with improvement of colon histological grade. The treatment also decreased the TNF-α, IFN-γ, IL-17 and IL-6 levels in colon. Furthermore, the treatment up-regulated the mRNA expression of CD25, Foxp3 and TGF-ß gene as well as the percentage of CD25+Foxp3+ T cells in colon lamina propria mononuclear cells (LPMCs) of IL-10-/- mice. Our data might indicate that anti-CD52 treatment could ameliorate the colitis of C3H.IL-10-/- mice and it might be related to the suppression of Th1/17 related inflammation and the promotion of regulatory T cell differentiation. Thus, our data reveals that anti-CD52 treatment may hold potential for clinical applications for Crohn's disease treatment.

Differential transcriptome of tolerogenic versus inflammatory dendritic cells points to modulated T1D genetic risk and enriched immune regulation.

Tolerogenic dendritic cells (tolDCs) are assessed as immunomodulatory adjuvants to regulate autoimmunity. The underlying gene expression endorsing their regulatory features remains ill-defined. Using deep mRNA sequencing, we compared transcriptomes of 1,25-dihydroxyvitaminD3/dexametasone-modulated tolDCs with that of non-modulated mature inflammatory DCs (mDCs). Differentially expressed genes controlled cellular interactions, metabolic pathways and endorse tolDCs with the capacity to regulate cell activation through nutrient and signal deprivation, collectively gearing tolDCs into tolerogenic immune regulators. Gene expression differences correlated with protein expression, designating low CD86 and high CD52 on the cell surface as superior discriminators between tolDCs and mDCs. Of 37 candidate genes conferring risk to developing type 1 diabetes (T1D), 11 genes differentially expressed in tolDCs and mDCs regulated immune response and antigen-presenting activity. Differential-expressed transcripts of candidate risk loci for T1D suggest a role of these 'risk genes' in immune regulation, which targeting may modulate the genetic contribution to autoimmunity.

Regenerative Potential of Spermatogonial Stem Cells, Endothelial Progenitor Cells, and Epithelial Progenitor Cells of C57Bl/6 Male Mice with Metabolic Disorders.

The properties of spermatogonial stem cells, endothelial progenitor cells, and the epithelial progenitors of C57Bl/6 mice under conditions of metabolic disorders were studied using the model of busulfan-induced suppression of spermatogenesis and in vitro culture technique. Spermatogonial stem cells CD117-CD90+ and epithelial progenitors CD45-CD31-Sca-1+CD49f+ derived from the testes of mice with metabolic disturbances demonstrated 17- and 28-fold increase in the respective cell mass and generated cell colonies in vitro. In contrast, spermatogonial stem cells with immune phenotype CD51-CD24+CD52+ had reduced selfrenewal capacity. Spermatogonial stem cells CD117-CD90+ and CD117+CD90+ as well as endothelial progenitors CD45-CD31+ derived from the testes of donor mice with metabolic disorders demonstrated high transplantation capacity in C57Bl/6 mouse testes damaged by cytostatic busulfan.

Isolation of CD63-positive epididymosomes from human semen and its application in improving sperm function.

Extracellular vesicles (EVs) are highly heterogeneous, and different EV subpopulations from various origins mediate different biological effects. The separation of different subpopulations of EVs from mixtures is critical but challenging. Epididymosomes are secreted by the epididymal epithelium and play a key role in sperm maturation and function. However, limited access to human epididymal tissue and epididymal fluid has hampered further study of epididymosomes and their potential clinical applications. Here, we established a novel strategy based on flow cytometry sorting to isolate human CD63-positive epididymosomes from ejaculate. We identified CD52, a membrane-located protein expressed exclusively in the epididymis, as the sorting marker for human epididymosomes. Then, CD63-positive epididymosomes were isolated from human semen using a flow cytometry sorting instrument and concentrated. Additionally, we observed that isolated CD63-positive epididymosomes improved sperm function more than other CD63-positive seminal EV subpopulations did, demonstrating the successful isolation of a subpopulation of epididymosomes from human semen and their potential application in the clinic.

Surface CD52, CD84, and PTGER2 mark mature PMN-MDSCs from cancer patients and G-CSF-treated donors.

Precise molecular characterization of circulating polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) is hampered by their mixed composition of mature and immature cells and lack of specific markers. Here, we focus on mature CD66b+CD10+CD16+CD11b+ PMN-MDSCs (mPMN-MDSCs) from either cancer patients or healthy donors receiving G-CSF for stem cell mobilization (GDs). By RNA sequencing (RNA-seq) experiments, we report the identification of a distinct gene signature shared by the different mPMN-MDSC populations under investigation, also validated in mPMN-MDSCs from GDs and tumor-associated neutrophils (TANs) by single-cell RNA-seq (scRNA-seq) experiments. Analysis of such a gene signature uncovers a specific transcriptional program associated with mPMN-MDSC differentiation and allows us to identify that, in patients with either solid or hematologic tumors and in GDs, CD52, CD84, and prostaglandin E receptor 2 (PTGER2) represent potential mPMN-MDSC-associated markers. Altogether, our findings indicate that mature PMN-MDSCs distinctively undergo specific reprogramming during differentiation and lay the groundwork for selective immunomonitoring, and eventually targeting, of mature PMN-MDSCs.

T-cell dysfunction in CLL is mediated through expression of Siglec-10 ligands CD24 and CD52 on CLL cells.

ABSTRACT: Autologous T-cell-based therapies, such as chimeric antigen receptor (CAR) T-cell therapy, exhibit low success rates in chronic lymphocytic leukemia (CLL) and correlate with a dysfunctional T-cell phenotype observed in patients. Despite various proposed mechanisms of T-cell dysfunction in CLL, the specific CLL-derived factors responsible remain unidentified. This study aimed to investigate the mechanisms through which CLL cells suppress CAR T-cell activation and function. We found that CLL-derived T cells get activated, albeit in a delayed fashion, and specifically that restimulation of CAR T cells in the presence of CLL cells causes impaired cytokine production and reduced proliferation. Notably, coculture of T cells with CD40-activated CLL cells did not lead to T-cell dysfunction, and this required direct cell contact between the CD40-stimulated CLL cells and T cells. Inhibition of kinases involved in the CD40 signaling cascade revealed that the Spare Respiratory Capacity (SRC) kinase inhibitor dasatinib prevented rescue of T-cell function independent of CD40-mediated increased levels of costimulatory and adhesion ligands on CLL cells. Transcriptome profiling of CD40-stimulated CLL cells with or without dasatinib identified widespread differential gene expression. Selecting for surface receptor genes revealed CD40-mediated downregulation of the Sialic acid-binding Ig-like lectin 10 (Siglec-10) ligands CD24 and CD52, which was prevented by dasatinib, suggesting a role for these ligands in functional T-cell suppression in CLL. Indeed, blocking CD24 and/or CD52 markedly reduced CAR T-cell dysfunction upon coculture with resting CLL cells. These results demonstrated that T cells derived from CLL patients can be reinvigorated by manipulating CLL-T-cell interactions. Targeting CD24- and CD52-mediated CLL-T-cell interaction could be a promising therapeutic strategy to enhance T-cell function in CLL.

Cis-interaction between CD52 and T cell receptor complex interferes with CD4+ T cell activation in acute decompensation of cirrhosis.

BACKGROUND: Immune dysfunction contributes to a high rate of infection in patients with acute decompensation of cirrhosis. CD52 is a glycoprotein prominently expressed in lymphocytes. Immune regulation by CD52 may be involved in adaptive immune dysfunction in cirrhosis. This study aimed to investigate the function of CD52 on CD4+ T cells on the blood of patients with acute decompensation of cirrhosis. METHODS: The expression of CD52 in the peripheral blood lymphocytes of 49 patients with cirrhosis was investigated using flow cytometry and transcriptomics. Potential cis-membrane ligands of CD52 were discovered via proximity labelling followed by proteomics. The function of CD52 on antigen-specific activation of CD4+ T cells was examined using flow cytometry in CD52 CRISPR-Cas9 knockout primary T cells. FINDINGS: CD52 expression was elevated in CD4+ T cells in acute decompensation of cirrhosis, and this elevation was correlated with increased disease severity and mortality. Components of the T cell receptor complex including TCRß, CD3γ and CD3ε were identified and validated as cis-membrane ligands of CD52. Knockout of CD52 promoted antigen-specific activation, proliferation, and pro-inflammatory cytokine secretion. INTERPRETATION: Membrane bound CD52 demonstrated cis-interaction with the T cell receptor and served as a dynamic regulator of antigen-specific activation of CD4+ T cells. The upregulation of CD52 in the periphery of acute decompensation of cirrhosis hinders the recognition of the T cell receptor by MHC, contributing to impaired T cell function. The development of an alternative anti-CD52 antibody is required to restore T cell function and prevent infections in cirrhosis. FUNDING: This study was supported by the NIHR Imperial Biomedical Research Centre, Institute for Translational Medicine and Therapeutics (P74713), Wellcome Trust (218304/Z/19/Z), and Medical Research Council (MR/X009904/1 and MR/R014019/1).

Interleukin-15 augments NK cell-mediated ADCC of alemtuzumab in patients with CD52+ T-cell malignancies.

Interleukin-15 (IL-15) monotherapy substantially increases the number and activity of natural killer (NK) cells and CD8+ T cells but has not produced clinical responses. In a xenograft mouse model, IL-15 enhanced the NK cell-mediated antibody-dependent cell cytotoxicity (ADCC) of the anti-CD52 antibody alemtuzumab and led to significantly more durable responses than alemtuzumab alone. To evaluate whether IL-15 potentiates ADCC in humans, we conducted a phase 1 single-center study of recombinant human IL-15 and alemtuzumab in patients with CD52-positive mature T-cell malignances. We gave IL-15 subcutaneously 5 days per week for 2 weeks in a 3 + 3 dose escalation scheme (at 0.5, 1, and 2 µg/kg), followed by standard 3 times weekly alemtuzumab IV for 4 weeks. There were no dose-limiting toxicities or severe adverse events attributable to IL-15 in the 11 patients treated. The most common adverse events were lymphopenia (100%), alemtuzumab-related infusion reactions (90%), anemia (90%), and neutropenia (72%). There were 3 partial and 2 complete responses, with an overall response rate of 45% and median duration of response 6 months. Immediately after 10 days of IL-15, there was a median 7.2-fold increase in NK cells and 2.5-fold increase in circulating CD8+ T cells, whereas the number of circulating leukemic cells decreased by a median 38% across all dose levels. Treatment with IL-15 was associated with increased expression of NKp46 and NKG2D, markers of NK-cell activation, and increased ex vivo ADCC activity of NK cells, whereas inhibitory receptors PD1 and Tim3 were decreased. This trial was registered at www.clinicaltrials.gov as #NCT02689453.

Regulation of Monocyte Adhesion and Type I Interferon Signaling by CD52 in Patients With Systemic Sclerosis.

OBJECTIVE: Systemic sclerosis (SSc) is characterized by dysregulation of type I interferon (IFN) signaling. CD52 is known for its immunosuppressive functions in T cells. This study was undertaken to investigate the role of CD52 in monocyte adhesion and type I IFN signaling in patients with SSc. METHODS: Transcriptome profiles of circulating CD14+ monocytes from patients with limited cutaneous SSc (lcSSc), patients with diffuse cutaneous SSc (dcSSs), and healthy controls were analyzed by RNA sequencing. Levels of CD52, CD11b/integrin αΜ, and CD18/integrin ß2 in whole blood were assessed by flow cytometry. CD52 expression was analyzed in relation to disease phenotype (early, lcSSc, dcSSc) and autoantibody profiles. The impact of overexpression, knockdown, and antibody blocking of CD52 was analyzed by gene and protein expression assays and functional assays. RESULTS: Pathway enrichment analysis indicated an increase in adhesion- and type I IFN-related genes in monocytes from SSc patients. These cells displayed up-regulated expression of CD11b/CD18, reduced expression of CD52, and enhanced adhesion to intercellular adhesion molecule 1 and endothelial cells. Changes in CD52 expression were consistent with the SSc subtypes, as well as with immunosuppressive treatments, autoantibody profiles, and monocyte adhesion properties in patients with SSc. Overexpression of CD52 led to decreased levels of CD18 and monocyte adhesion, while knockdown of CD52 increased monocyte adhesion. Experiments with the humanized anti-CD52 monoclonal antibody alemtuzumab in blood samples from healthy controls increased monocyte adhesion and CD11b/CD18 expression, and enhanced type I IFN responses. Monocytic CD52 expression was up-regulated by interleukin-4 (IL-4)/IL-13 via the STAT6 pathway, and was down-regulated by lipopolysaccharide and IFNs α, ß, and γ in a JAK1 and histone deacetylase IIa (HDAC IIa)-dependent manner. CONCLUSION: Down-regulation of the antiadhesion CD52 antigen in CD14+ monocytes represents a novel mechanism in the pathogenesis of SSc. Targeting of the IFN-HDAC-CD52 axis in monocytes might represent a new therapeutic option for patients with early SSc.

Alemtuzumab: A Review in Relapsing Remitting Multiple Sclerosis.

Alemtuzumab (Lemtrada®) is an anti-CD52 monoclonal antibody approved in the EU for the treatment of highly active relapsing-remitting multiple sclerosis (RRMS). In phase 3 trials in patients with active RRMS, intravenous alemtuzumab was more effective than subcutaneous interferon ß-1a in terms of decreasing relapse rates (in treatment-naïve or -experienced patients) and disability progression (treatment-experienced patients). Treatment benefits were maintained over up to 9 years of follow-up, with ≈ 50% of patients not requiring retreatment. The efficacy of alemtuzumab in patients with highly active disease was generally similar to that in the overall population. Alemtuzumab has an acceptable tolerability profile, with infusion-associated reactions, infections and autoimmunity being the main safety and tolerability issues. Current evidence indicates that alemtuzumab is an effective treatment option for adults with highly active RRMS, with an acceptable safety and tolerability profile and convenient treatment regimen.

High expression of CD52 in adipocytes: a potential therapeutic target for obesity with type 2 diabetes.

The aim of the present study was to evaluate the involvement of CD52 in adipocytes as well as to explore its effect on type 2 diabetes mellitus (T2DM), and to improve our understanding of the potential molecular events of obesity with type 2 diabetes. Global changes in the CD52 expression patterns were detected in adipocytes and preadipocytes derived from obese and lean individuals. In particular, CD52 was identified as significantly differentially upregulated and was analyzed, both in vitro and in vivo, using various approaches. In vitro experiments, CD52 was a significantly up-regulated mRNA in mature adipocytes and preadipocytes. In addition, CD52 gradually increased with the differentiation of preadipocytes. In vivo experiments, the expression of CD52 in high-fat diet (HFD) -fed mice tended to be higher than that in regular diet (RD) -fed mice. Further analysis showed that CD52 expression was positively correlated with Smad3 and TGF-ß in mice, and the downregulation of CD52 was accompanied by increased glucose tolerance and insulin sensitivity. Moreover, a comparison of CD4+CD52high T cells and CD4+CD52low T cells showed that many T2DM-related genes were aberrantly expressed. Overall, CD52 may functioned as an important potential target for obesity with T2DM via TGF-ß/Smad3 axis.

Immune Regulatory Cell Bias Following Alemtuzumab Treatment in Relapsing-Remitting Multiple Sclerosis.

Alemtuzumab is a highly effective treatment for relapsing-remitting multiple sclerosis. It selectively targets the CD52 antigen to induce profound lymphocyte depletion, followed by recovery of T and B cells with regulatory phenotypes. We previously showed that regulatory T cell function is restored with cellular repletion, but little is known about the functional capacity of regulatory B-cells and peripheral blood monocytes during the repletion phase. In this study (ClinicalTrials.gov ID# NCT03647722) we simultaneously analyzed the change in composition and function of both regulatory lymphocyte populations and distinct monocyte subsets in cross-sectional cohorts of MS patients prior to or 6, 12, 18, 24 or 36 months after their first course of alemtuzumab treatment. We found that the absolute number and percentage of cells with a regulatory B cell phenotype were significantly higher after treatment and were positivity correlated with regulatory T cells. In addition, B cells from treated patients secreted higher levels of IL-10 and BDNF, and inhibited the proliferation of autologous CD4+CD25- T cell targets. Though there was little change in monocytes populations overall, following the second annual course of treatment, CD14+ monocytes had a significantly increased anti-inflammatory bias in cytokine secretion patterns. These results confirmed that the immune system in alemtuzumab-treated patients is altered in favor of a regulatory milieu that involves expansion and increased functionality of multiple regulatory populations including B cells, T cells and monocytes. Here, we showed for the first time that functionally competent regulatory B cells re-appear with similar kinetics to that of regulatory T-cells, whereas the change in anti-inflammatory bias of monocytes does not occur until after the second treatment course. These findings justify future studies of all regulatory cell types following alemtuzumab treatment to reveal further insights into mechanisms of drug action, and to identify key immunological predictors of durable clinical efficacy in alemtuzumab-treated patients.

Treatment With CD52 Antibody Protects Neurons in Experimental Autoimmune Encephalomyelitis Mice During the Recovering Phase.

Multiple sclerosis (MS) is a chronic autoimmune disease driven by T and B lymphocytes. The remyelination failure and neurodegeneration results in permanent clinical disability in MS patients. A desirable therapy should not only modulate the immune system, but also promote neuroprotection and remyelination. To investigate the neuroprotective effect of CD52 antibody in MS, both C57BL/6J and SJL mice with experimental autoimmune encephalomyelitis (EAE) were treated with CD52 antibody at the peak of disease. Treatment with CD52 antibody depleted T but not B lymphocytes in the blood, reduced the infiltration of T lymphocytes and microglia/macrophages in the spinal cord. Anti-CD52 therapy attenuated EAE scores during the recovery phase. It protected neurons immediately after treatment (within 4 days) as shown by reducing the accumulation of amyloid precursor proteins. It potentially promoted remyelination as it increased the number of olig2/CC-1-positive mature oligodendrocytes and prevented myelin loss in the following days (e.g., 14 days post treatment). In further experiments, EAE mice with a conditional knockout of BDNF in neurons were administered with CD52 antibodies. Neuronal deficiency of BDNF attenuated the effect of anti-CD52 treatment on reducing EAE scores and inflammatory infiltration but did not affect anti-CD52 treatment-induced improvement of myelin coverage in the spinal cord. In summary, anti-CD52 therapy depletes CD4-positive T lymphocytes, prevents myelin loss and protects neurons in EAE mice. Neuronal BDNF regulates neuroprotective and anti-inflammatory effect of CD52 antibody in EAE mice.