Immunomodulatory effects of intravenous and subcutaneous immunoglobulin in chronic inflammatory demyelinating polyneuropathy: An observational study.

BACKGROUND AND PURPOSE: It is not known whether the route of administration affects the mechanisms of action of therapeutic immunoglobulin in chronic inflammatory demyelinating polyneuropathy (CIDP). The aim of this study, therefore, was to compare the immunomodulatory effects of intravenous (IVIg) and subcutaneous immunoglobulin (SCIg) in patients with CIDP and in IVIg-treated common variable immunodeficiency (CVID) patients. METHODS: Serum and peripheral blood mononuclear cell samples were obtained from 30 CIDP patients receiving IVIg, 10 CIDP patients receiving SCIg, and 15 patients with CVID receiving IVIg. Samples and clinical data were obtained prior to IVIg/SCIg and at 3 days, 7 days, and, in CIDP patients receiving IVIg, 21 days post-administration. Serum cytokines were assessed by Luminex-based multiplex assay and enzyme-linked immunosorbent assay. Immune cells were characterized by flow cytometry. RESULTS: Immune cell profiles of CIDP and CVID patients differed in frequencies of myeloid dendritic cells and cytotoxic natural killer cells. During treatment with IVIg or SCIg in CIDP patients, cellular immunomarkers were largely similar. CIDP patients receiving IVIg had higher macrophage inflammatory protein (MIP)-1α (p = 0.01), interleukin (IL)-4 (p = 0.04), and IL-33 (p = 0.04) levels than SCIg recipients. IVIg treatment more broadly modulated cytokines in CIDP than SCIg treatment. CONCLUSIONS: Our study demonstrates that the modulation of cellular immunomarkers in CIDP is independent of the application route of therapeutic immunoglobulin. Minor differences were observed between CIDP and CVID patients. In contrast, cytokines were differentially modulated by IVIg and SCIg in CIDP.

The Immunosuppressive Receptor CD32b Regulation of Macrophage Polarization and Its Implications in Tumor Progression.

Macrophages, pivotal components of the immune system, orchestrate host defense mechanisms in humans and mammals. Their polarization into classically activated macrophages (CAMs or M1) and alternatively activated macrophages (AAMs or M2) dictates distinct functional roles in immunity and tissue homeostasis. While the negative regulatory role of CD32b within the FC gamma receptor (FCγR) family is recognized across various immune cell types, its influence on macrophage polarization remains elusive. This study aimed to elucidate the regulatory role of CD32b in macrophage polarization and discern the differential expression markers between the M1 and M2 phenotypes following CD32b siRNA transfection. The results revealed a decrease in the CD32b levels in lipopolysaccharide (LPS)-treated M1 and an increase in interleukin-4 (IL-4)-treated M2 macrophages, as observed in macrophage Raw264.7 cells. Furthermore, CD32b siRNA transfection significantly downregulated the M2 markers (IL-10, VEGF, Arg-1, and STAT6), while upregulating the M1 markers (IL-6, NF-κB, NOS2, and STAT1) in the Raw264.7 cells. Similar findings were recapitulated in macrophage-rich adherent cells isolated from mouse spleens. Additionally, the cytopathological analysis of pleural effusions and ascitic fluids from patients with cancer revealed a positive correlation between advanced tumor stages, metastasis, and elevated CD32b levels. In conclusion, this study highlights the regulatory influence of CD32b in suppressing M1 expression and promoting M2 polarization. Moreover, heightened M2 activation and CD32b levels appear to correlate with tumor progression. A targeted CD32b blockade may serve as a novel therapeutic strategy to inhibit M2 macrophage polarization and is promising for anti-tumor intervention.

Down-regulated FcγRII expression on plasma cells is associated with the disease activity of ANCA-associated vasculitis.

OBJECTIVES: Inhibitory FcγRIIB/CD32B on B cells are critical for immunity regulation to help maintain peripheral tolerance. Altered FcγRIIB expression on B cells has been observed in several autoimmune diseases, and animal studies have suggested that FcγRIIB on B cells participates in the pathogenesis of ANCA-associated vasculitis (AAV). Here, we investigated the expression of FcγRII (FcγRIIB) on various B cell subsets and the correlation of FcγRII/CD32 expression with disease activity in AAV patients. MATERIAL AND METHODS: Blood samples of patients with AAV in active stage and in remission were collected. FcγRII/CD32 expressions on various B cell subsets of the whole blood were detected by flow cytometry, and their correlation with clinical and pathological data was analysed. RESULTS: The expression of FcγRII/CD32 on plasma cells was significantly lower in AAV patients in active stage than those in both AAV patients in remission and healthy donors. Furthermore, the expression of FcγRII/CD32 on plasma cells negatively correlated with BVAS and percentages of cellular crescents in renal biopsies. CONCLUSIONS: There is a down-regulation of FcγRIIB/CD32B expression on B cells in patients with AAV, which is associated with the disease activity of AAV.

Decreased expression of FcγRII in active Graves' disease patients.

BACKGROUND: Graves' disease (GD) is a common autoimmune disease characterized by genetic and environmental factors. Fcγ receptors (FcγRs) are involved in several autoimmune disorders through recognizing immunoglobulin (Ig) G antibodies and mediating immune response. The study on the expression of FcγRs in GD patients is scarce. The purpose of this study was to evaluate the expression of three different types of FcγRs in patients with active and remissive GD. METHODS: Blood samples of patients and healthy subjects were collected to analyze the percentage of FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16) on peripheral blood mononuclear cells (PBMCs) and monocytes by flow cytometry and Western blotting. CD32 isotypes were also examined in cases and controls by real-time PCR. RESULTS: The cell percentages expressed CD32 and protein expressions of CD32 on PBMCs, and monocytes from patients with active GD were significantly reduced compared to controls and patients with remissive GD. In particular, the expression of CD32B on PBMC was also decreased in active GD patients. However, the cell percentages expressed CD16 and CD64 from PBMCs and monocytes were comparable between three groups. Besides, the percentages of CD14+ CD32+ cells were negatively correlated with TRAb titers in active GD patients (r = -0.5825, P ï¼œ 0.001). CONCLUSION: These results suggested that CD32 may act as a novel marker for active GDs. The expression of monocytic CD32, in particular CD32B, in GD patients might play a crucial role in maintaining FcγRs function and be a therapeutic target in GD patients.

Regulation of Monoclonal Antibody Immunotherapy by FcγRIIB.

Monoclonal antibodies (mAb) are revolutionising the treatment of many different diseases. Given their differing mode of action compared to most conventional chemotherapeutics and small molecule inhibitors, they possess the potential to be independent of common modes of treatment resistance and can typically be combined readily with existing treatments without dose-limiting toxicity. However, treatments with mAb rarely result in cure and so a full understanding of how these reagents work and can be optimised is key for their subsequent improvement. Here we review how an understanding of the biology of the inhibitory Fc receptor, FcγRIIB (CD32B), is leading to the development of improved mAb treatments.

The CD16 and CD32b Fc-gamma receptors regulate antibody-mediated responses in mouse natural killer cells.

Natural killer (NK) cells are innate lymphocytes capable of mediating immune responses without prior sensitization. NK cells express Fc-gamma receptors (FcγRs) that engage the Fc region of IgG. Studies investigating the role of FcγRs on mouse NK cells have been limited due to lack specific reagents. In this study, we characterize the expression and biological consequences of activating mouse NK cells through their FcγRs. We demonstrate that most NK cells express the activating CD16 receptor, and a subset of NK cells also expresses the inhibitory CD32b receptor. Critically, these FcγRs are functional on mouse NK cells and can modulate antibody-mediated responses. We also characterized mice with conditional knockout alleles of Fcgr3 (CD16) or Fcgr2b (CD32b) in the NK and innate lymphoid cell (ILC) lineage. NK cells in these mice did not reveal any developmental defects and were responsive to cross-linking activating NK receptors, cytokine stimulation, and killing of YAC-1 targets. Importantly, CD16-deficient NK cells failed to induce antibody-directed cellular cytotoxicity of antibody-coated B-cell lymphomas in in vitro assays. In addition, we demonstrate the important role of CD16 on NK cells using an in vivo model of cancer immunotherapy using anti-CD20 antibody treatment of B-cell lymphomas.

CIDP: Analysis of Immunomarkers During COVID-19 mRNA-Vaccination and IVIg-Immunomodulation: An Exploratory Study.

Availability of COVID-19 mRNA vaccine for patients with chronic inflammatory demyelinating polyneuropathy (CIDP) treated with intravenous immunoglobulin (IVIg) raises the question of whether COVID-19 mRNA vaccine influences disease activity or IVIg-mediated immunomodulation in CIDP. In this exploratory study, blood samples of CIDP patients on IVIg treatment were longitudinally analyzed before and after vaccination with a COVID-19 mRNA vaccine. A total of 44 samples of eleven patients were characterized at four timepoints by ELISA and flow cytometry in terms of immunomarkers for disease activity and IVIg-immunomodulation. Apart from a significantly lower expression of CD32b on naïve B cells after vaccination, no significant alteration of immunomarkers for CIDP or IVIg-mediated immunomodulation was observed. Our exploratory study suggests that COVID-19 mRNA vaccine does not have a relevant impact on immune activity in CIDP. In addition, immunomodulatory effects of IVIg in CIDP are not altered by COVID-19 mRNA vaccine. This study was registered in the German clinical trial register (DRKS00025759). Overview over the study design. Blood samples of CIDP patients on recurrent IVIg treatment and vaccination with a COVID-19 mRNA vaccine were obtained at four timepoints for cytokine ELISA and flow cytometry, to assess key cytokines and cellular immunomarkers for disease activity and IVIg-immunomodulation in CIDP.

Aberrant expression of inhibitory receptors on B cells in patients with Graves' disease.

The pathophysiological mechanism underlying Graves' disease (GD) remains incompletely understood. Inhibitory receptors on B cells are critical for humoral immunity, which plays a key role in GD pathogenesis. This study aimed to investigate B cell subsets distribution and inhibitory receptor expression on these subsets in GD patients. Peripheral blood was drawn from 41 healthy controls and 46 GD patients (21 patients with moderate GD, 25 patients with severe GD). B cell subset distribution and CD22, CD32b and CD72 expression on B cells were analyzed by flow cytometry. Serum cytokines were examined by enzyme-linked immunosorbent assay (ELISA). Compared with healthy controls, the naïve B cell percentage was increased, while the preswitched memory and conventional memory B cell percentages were decreased. The inhibitory receptors expression, especially CD32b, on B cell subsets was significantly decreased in patients with GD. In addition, the inhibitory receptors expression on B cell subsets from severe GD patients exhibited a decreasing trend compared with those from moderate GD patients. These results suggest that abnormal B cell subset distribution occurs in GD. Impaired inhibitory receptors, in particular CD32b, play a crucial role in GD pathogenesis and might be a therapeutic target to rebuild self-immune tolerance in GD.

Homogeneously high expression of CD32b makes it a potential target for CAR-T therapy for chronic lymphocytic leukemia.

CD19 chimeric antigen receptor (CAR)-T cells have been used to treat patients with refractory chronic lymphocytic leukemia (CLL). However, approximately 50% of patients do not respond to this therapy. To improve the clinical outcome of these patients, it is necessary to develop strategies with other optimal targets to enable secondary or combinational CAR-T cell therapy. By screening a panel of surface antigens, we found that CD32b (FcγRIIb) was homogeneously expressed at high site density on tumor cells from CLL patients. We then developed a second-generation CAR construct targeting CD32b, and T cells transduced with the CD32 CAR efficiently eliminated the CD32b+ Raji leukemic cell line in vitro and in a mouse xenograft model. Furthermore, CD32b CAR-T cells showed cytotoxicity against primary human CLL cells that were cultured in vitro or transplanted into immunodeficient mice. The efficacy of CD32b CAR T cells correlated with the CD32b density on CLL cells. CD32b is not significantly expressed by non-B hematopoietic cells. Our study thus identifies CD32b as a potential target of CAR-T cell therapy for CLL, although further modification of the CAR construct with a safety mechanism may be required to minimize off-target toxicity.

Fc-gamma receptor expression profile in a North-Indian cohort of pediatric-onset systemic lupus erythematosus: An observational study.

BACKGROUND: Polymorphisms in the Fcγ-receptor (FcγR) have been associated with increased susceptibility to systemic lupus erythematosus (SLE). There is a paucity of data on FcγR expression pattern in pediatric subjects with SLE. The aim of the study was to assess the expression of various FcγRs by flow cytometry in children with pediatric-onset SLE (pSLE). METHODS: Thirty-one children aged 0-15 years fulfilling 2012 Systemic Lupus International Collaborating Clinics Classification Criteria for SLE were enrolled. Disease-active (n = 14) and the inactive group were delineated using the SLE Disease Activity Index (SLEDAI). Thirteen age- and sex-matched controls were also enrolled. Blood samples of cases and controls were assessed for CD64, CD32B and CD16 expression on B lymphocytes, neutrophils and monocytes by flow cytometry using standard techniques. Median fluorescence intensity (MFI) and percentage expression were calculated using the FACS DIVA software and Kaluza software. RESULTS: Median fluorescence intensity and percentage expression of CD64 on monocytes (MFI: 1.71 vs 1.51, P = 0.86) and neutrophils (MFI: 0.42 vs 0.64, P = 0.3) were comparable between patients and controls. MFIs of CD16 expression on neutrophils (3.47 vs 11.4, P = 0.05) and monocytes (1.28 vs 3.45, P = 0.07) were lower in patients compared to controls. CD32B expression on lymphocytes (MFI: 0.56 vs 1.37; % expression:18.3% vs 12.32%) was also comparable between cases and controls. Expression of CD64, CD16, and CD32B were also comparable between patients with active and inactive disease. CONCLUSION: No significant differences were observed in FcγR expression between patients and controls. However, the overall trends of FcγR expression and decreased CD16 on monocytes and neutrophils are in consonance with data from larger cohorts of adult SLE patients.

CD32b expression is down-regulated on double-negative memory B cells in patients with Hashimoto's thyroiditis.

Inhibitory CD32b receptors on B cells are critical for humoral immunity. The humoral response plays a role in the pathogenesis of Hashimoto's thyroiditis (HT). This study aimed to investigate B cell subset distribution and CD32b expression within these subsets in HT patients. B cell subset distribution and CD32b expression were analyzed in 60 HT patients and 21 healthy donors. Subset distribution and CD32b expression following stimulation with α-Ig and α-CD40 were also assessed. The percentage of double-negative (DN) memory cells was increased in the HT patients, while the expression level of CD32b on DN memory cells was decreased. Redistribution of B cell subsets was detected in response to stimulation with α-Ig. In addition, the expression level of CD32b was reduced following α-CD40 stimulation. These results suggest that abnormal B cell subset distribution and decreased CD32b expression on DN memory cells might be involved in the pathogenesis of HT.

Resistance is futile: Targeting the inhibitory FcγRIIB (CD32B) to maximize immunotherapy.

Monoclonal antibodies (mAb) are central to the treatment of several types of malignancy. However, these reagents are subject to particular types of resistance. Several resistance mechanisms are regulated by the inhibitory FcγRIIB. We recently developed mAbs to block FcγRIIB and provided in vivo proof-of-concept for their ability to overcome FcγRIIB-mediated resistance.

Engagement of CD22 on B cells with the monoclonal antibody epratuzumab stimulates the phosphorylation of upstream inhibitory signals of the B cell receptor.

The binding of antigen to the B cell receptor (BCR) results in a cascade of signalling events that ultimately drive B cell activation. Uncontrolled B cell activation is regulated by negative feedback loops that involve inhibitory co-receptors such as CD22 and CD32B that exert their functions following phosphorylation of immunoreceptor tyrosine-based inhibition motifs (ITIMs). The CD22-targeted antibody epratuzumab has previously been shown to inhibit BCR-driven signalling events, but its effects on ITIM phosphorylation of CD22 and CD32B have not been properly evaluated. The present study therefore employed both immunoprecipitation and flow cytometry approaches to elucidate the effects of epratuzumab on direct phosphorylation of key tyrosine (Tyr) residues on both these proteins, using both transformed B cell lines and primary human B cells. Epratuzumab induced the phosphorylation of Tyr(822) on CD22 and enhanced its co-localisation with SHP-1. Additionally, in spite of high basal phosphorylation of other key ITIMs on CD22, in primary human B cells epratuzumab also enhanced phosphorylation of Tyr(807), a residue involved in the recruitment of Grb2. Such initiation events could explain the effects of epratuzumab on downstream signalling in B cells. Finally, we were able to demonstrate that epratuzumab stimulated the phosphorylation of Tyr(292) on the low affinity inhibitory Fc receptor CD32B which would further attenuate BCR-induced signalling. Together, these data demonstrate that engagement of CD22 with epratuzumab leads to the direct phosphorylation of key upstream inhibitory receptors of BCR signalling and may help to explain how this antibody modulates B cell function.

Immunohistochemical detection of CD14 and combined assessment with CD32B and CD68 for wound age estimation.

Estimation of wound age is a major topic of study for forensic pathologists, but few markers exist that can indicate a specific period 1-5 days postinfliction, and a method to estimate wound age with high accuracy has not yet been established. This study examined CD14 as such a marker in mouse skin wounds of different ages (0min and 1, 2, 3, 5, 7, and 9 days) and in human subjects (group 1, 0-1 day; group 2, 1-5 days; group 3, >7 days) using Western blot analysis and/or immunohistochemical staining. In addition, we evaluated a combination of immunohistochemical markers in human skin wounds using transmembrane proteins, CD14, CD32B, and CD68, expressed on inflammatory cells. The expression of CD14 was detected only during 1-5 days postinfliction and, thus, the evaluation of CD14-expressing cells could specify wound age during 1-5 days postinfliction in mouse skin wounds. The ratio of samples assessed to be CD14(+) was significantly high in human skin wounds in group 2. Combined assessment using the three markers increased the specificity of diagnosis and shortened the range of wound age, compared with the assessment using a single marker. Our results indicate that CD14 may be a useful marker of wound age, 1-5 days postinfliction, and that combined assessment with CD14, CD32B, and CD68 may be a good method for the accurate estimation of wound age.