B-cell Research Articles

Evolution of pathologic B-cell subsets and serum environment-specific sIgEs in patients with atopic dermatitis and controls, from infancy to adulthood.

While B-cells have historically been implicated in allergy development, a growing body of evidence supports their role in atopic dermatitis (AD). B-cell differentiation across ages in AD, and its relation to disease severity scores, has not been well defined. To compare the frequency of B-cell subsets in blood of 0-5, 6-11, 12-17, and ≥18 years old patients with AD versus age-matched controls. Flow cytometry was used to measure B-cell subset frequencies in the blood of 27 infants, 17 children, 11 adolescents, and 31 adults with moderate-to-severe AD and age-matched controls. IgD/CD27 and CD24/CD38 core gating systems and an 11-color flow cytometry panel were used to determine frequencies of circulating B-cell subsets. Serum total and allergen-specific IgE (sIgEs) levels were measured using ImmunoCAP®. Adolescents with AD had lower frequencies of major B-cells subsets (p < .03). CD23 expression increased with age and was higher in AD compared to controls across all age groups (p < .04). In AD patients, multiple positive correlations were observed between IL-17-producing T-cells and B-cell subsets, most significantly non-switched memory (NSM) B-cells (r = .41, p = .0005). AD severity positively correlated with a list of B-cell subsets (p < .05). IL-9 levels gradually increased during childhood, reaching a peak in adolescence, paralleling allergen sensitization, particularly in severe AD. Principal component analysis of the aggregated environmental sIgE data showed that while controls across all ages tightly clustered together, adolescents with AD demonstrated distinct clustering patterns relative to controls. Multiple correlations between B-cells and T-cells, as well as disease severity measures, suggest a complex interplay of immune pathways in AD. Unique B-cell signature during adolescence, with concurrent allergen sensitization and IL-9 surge, point to a potentially wider window of opportunity to implement interventions that may prevent the progression of the atopic march.

Phagocytic Plasma Cells in Teleost Fish Provide Insights into the Origin and Evolution of B Cells in Vertebrates.

Teleost IgM+ B cells can phagocytose, like mammalian B1 cells, and secrete Ag-specific IgM, like mammalian B2 cells. Therefore, teleost IgM+ B cells may have the functions of both mammalian B1 and B2 cells. To support this view, we initially found that grass carp (Ctenopharyngodon idella) IgM+ plasma cells (PCs) exhibit robust phagocytic ability, akin to IgM+ naive B cells. Subsequently, we sorted grass carp IgM+ PCs into two subpopulations: nonphagocytic (Pha-IgM+ PCs) and phagocytic IgM+ PCs (Pha+IgM+ PCs), both of which demonstrated the capacity to secrete natural IgM with LPS and peptidoglycan binding capacity. Remarkably, following immunization of grass carp with an Ag, we observed that both Pha-IgM+ PCs and Pha+IgM+ PCs could secrete Ag-specific IgM. Furthermore, invitro concatenated phagocytosis experiments in which Pha-IgM+ PCs from an initial phagocytosis experiment were sorted and exposed again to beads confirmed that these cells also have phagocytic capabilities, thereby suggesting that all teleost IgM+ B cells have phagocytic potential. Additionally, we found that grass carp IgM+ PCs display classical phenotypic features of macrophages, providing support for the hypothesis that vertebrate B cells evolved from ancient phagocytes. These findings together reveal that teleost B cells are a primitive B cell type with functions reminiscent of both mammalian B1 and B2 cells, providing insights into the origin and evolution of B cells in vertebrates.

Application of peripheral blood cytokine and immunoglobulin detection in ACTH therapy for the treatment of infantile spasms.

This research aims to investigate the levels of lymphocytes, immunoglobulins, and cytokines in children with infantile spasms (IS) before and after adrenocorticotropic hormone (ACTH) therapy and to explore the application of these markers in evaluating the therapeutic effects of ACTH on infantile spasms. From May to November 2022, 35 children initially diagnosed with IS and treated at our hospital were regarded as the observation group, and 35 healthy children who underwent physical examination at our hospital during the same period were regarded as the control group. Children in the observation group received intramuscular injections of ACTH for 2 weeks. Fasting venous blood was collected from the control group and the observation group before and after ACTH therapy. Serum levels of immunoglobulins IgG, IgA, and IgM in serum were detected by immunoturbidimetry. T-cell subsets (CD3+, CD3+CD4+, and CD3+CD8+) and B-cell subsets [CD3-CD19+ and CD3-CD16+CD56+ natural killer (NK) cells] were detected by flow cytometry, and the ratio of CD3+CD4+/CD3+CD8+ was calculated. Serum levels of interleukin-1β (IL-1β), interleukin-2R (IL-2R), and interleukin-6 (IL-6) cytokines were detected by the enzyme-linked immunosorbent assay, and changes in serum cytokine and immunoglobulin levels in the two groups were compared before therapy, whereas in observation group one, these comparisons were made both before and after ACTH therapy. Compared to the control group, the observation group showed significantly increased serum levels of immunoglobulins IgG and IgM before therapy, while the level of IgA was significantly decreased (p < 0.05). Also, the percentage of CD3-CD19+ B cells was significantly increased, while the percentages of CD3+ T cells and CD3+CD4+ T cells were significantly decreased (p < 0.05). The percentages of CD3+CD8+ T cells, CD3-CD16+CD56+ NK cells, and CD3+CD4+/CD3+CD8+ cells did not change significantly (p > 0.05); the levels of cytokines IL-1 β, IL-2R, and IL-6 were significantly increased (p < 0.05). Compared to levels before treatment, the serum level of immunoglobulin IgG in the observation group after ACTH therapy was significantly reduced (p < 0.05), while the IgA and IgM levels did not change significantly (p > 0.05). The percentages of CD3+ T cells and CD3+CD4+ T cells were significantly increased, while the percentages of CD3-CD16+CD56+ NK cells and CD3-CD19+ B cells were significantly decreased (p < 0.05); however, the percentages of CD3+CD8+ T cells and the CD3+CD4+/CD3+CD8+ ratio did not change significantly (p > 0.05). Furthermore, the levels of cytokines IL-1 β, IL-2R, and IL-6 were significantly reduced (p < 0.05). Children with IS exhibit immune dysfunction, and the changes in serological immune indices after ACTH treatment indicate that ACTH may control seizures in IS children by regulating and improving immune dysfunction. Therefore, the therapeutic effects of ACTH on IS can be evaluated by detecting the levels of cytokines and immunoglobulins.

Neural Stem Cell Relay from B1 to B2 cells in the adult mouse Ventricular-Subventricular Zone.

Neurogenesis and gliogenesis continue in the Ventricular-Subventricular Zone (V-SVZ) of the adult rodent brain. B1 cells are astroglial cells derived from radial glia that function as primary progenitors or neural stem cells (NSCs) in the V-SVZ. B1 cells, which have a small apical contact with the ventricle, decline in numbers during early postnatal life, yet neurogenesis continues into adulthood. Here we found that a second population of V-SVZ astroglial cells (B2 cells), that do not contact the ventricle, function as NSCs in the adult brain. B2 cell numbers increase postnatally, remain constant in 12-month-old mice and decrease by 18 months. Transcriptomic analysis of ventricular-contacting and non-contacting B cells revealed key molecular differences to distinguish B1 from B2 cells. Transplantation and lineage tracing of B2 cells demonstrate their function as primary progenitors for adult neurogenesis. This study reveals how NSC function is relayed from B1 to B2 progenitors to maintain adult neurogenesis.

Glycolysis Changes in Alloreactive Memory B Cells in Highly Sensitized Kidney Transplant Recipients Undergonig Desensitization Therapy.

Despite the growing use of desensitization strategies, hyperimmune patients remain at high risk of antibody-mediated rejection suggesting that, even when donor-specific antibodies (DSA) are effectively depleted, anti-donor specific B cells persist. We included 10 highly sensitized recipients that underwent desensitization with plasmapheresis and B cell depletion prior to kidney transplantation. We quantified changes in DSA (luminex), total B-cell subsets (flow cytometry), anti-donor HLA B cells (fluorospot), and single-cell metabolism in serially collected samples before desensitization, at the time of transplant, and at 6 and 12months thereafter. Desensitization was associated with a decrease in DSA and total memory B cell and naive B cell percentage, while plasma cells and memory anti-donor HLA circulating B cells persisted up to 12months after transplant. At 12-month post-transplantation, memory B cells increased their glycolytic capacity, while proliferative KI67+ plasma cells modified their metabolism by increasing fatty acid and amino acid oxidation capacity and decreasing their glucose dependence. Despite effective DSA depletion, anti-donor B cells persist in kidney transplant recipients. Due to the reliance of these cells on glycolysis, glycolysis-targeting therapies might represent a valuable treatment strategy.

Sex as a predictor of clinical phenotype and determinant of immune response in IgG4-related disease: a retrospective study of patients fulfilling the American College of Rheumatology-European League Against Rheumatism classification criteria.

IgG4-related disease is a multiorgan fibroinflammatory disease considered to have an autoimmune origin. Case series describing individual organ involvement have suggested differences in phenotypic expression between males and females. We aimed to characterise differences in IgG4-related disease manifestations between male and female patients in a large single-centre cohort. In this retrospective, single-centre cohort study, patients were recruited from the Massachusetts General Hospital Rheumatology Clinic (Boston, MA, USA) and classified according to the American College of Rheumatology-European Alliance of Associations for Rheumatology (ACR-EULAR) classification criteria. Only patients satisfying the ACR-EULAR classification criteria were included in the study. Data on age at diagnosis, organ involvement at baseline, treatment status, and pre-treatment laboratory values were collected. Circulating plasmablasts and B-cell subsets were quantitated by flow cytometry. Active disease was defined by an IgG4-related disease Responder Index score of more than 0. Laboratory values were analysed for patients who were untreated at baseline and had active IgG4-related disease. The main outcomes were assessed in all participants with available data. Of the 564 participants enrolled in the Massachusetts General Hospital Rheumatology Clinic IgG4-related disease Registry, 328 fulfilled ACR-EULAR classification criteria and were included between January, 2008, and May, 2023. There was a strong male predominance (male:female ratio 2·2:1) with 226 (69%) males and 102 (31%) females, which contrasted markedly with our general rheumatology clinic population (0·4:1; p<0·001). The male predominance increased with each decade of life starting at age 40 years. On average, male patients were 5·5 years older at diagnosis than female patients (63·7 years vs 58·2 years; p=0·0031). We observed male patients to have higher ACR-EULAR classification criteria scores at baseline with a median score of 35·0 (IQR 28·0-46·0), compared with 29·5 (25·0-39·0) for females (p=0·0010). The proportion of male patients with pancreatic and renal involvement was almost double the proportion observed in female patients (50% of the male patients had pancreatic involvement, compared with about 26% of the female patients; p<0·0001). Male patients were more likely to have serological abnormalities at baseline. The distribution of IgG4 values differed significantly between male an female sexes, favouring higher values in males. We found that male patients with IgG4-related disease were more likely to have active B-cell responses in the blood as defined by plasmablast expansions. IgG4-related disease is unusual among autoimmune diseases in that it is more likely to affect males than females and to present with a striking sex-dependent organ distribution and degree of B-cell response. These findings highlight important variation between IgG4-related disease and other conditions generally believed to have an autoimmune basis. Most autoimmune diseases, by contrast to IgG4-related disease, demonstrate pronounced predilections for affecting females more frequently than males. Hypotheses surrounding the cause and pathophysiology of this condition need to consider this unusual sex distribution among patients with IgG4-related disease. National Institutes of Health, National Institute of Allergy and Infectious Diseases, Rheumatology Research Foundation, and the National Institute of Arthritis and Musculoskeletal and Skin Diseases.

Varied immune responses of HBV-specific B cells in patients undergoing pegylated interferon-alpha treatment for chronic hepatitis B

The changes in HBV-specific B cells in patients with chronic hepatitis B (CHB) undergoing pegylated interferon-α (PEG-IFNα) treatment and achieving functional cure remain unclear. We aimed to evaluate the alterations in HBV-specific B cells during treatment and therefore explored the mechanism of functional recovery of HBsAg-specific B cells. We included 39 nucleos(t)ide analogue-treated patients with CHB who received sequential combination therapy with PEG-IFNα and eight treatment-naïve patients. HBV-specific B cells were characterized exvivo using fluorescently labeled hepatitis B surface and core antigens (HBsAg and HBcAg). The frequency, phenotype, and subsets of HBV-specific B cells and follicular helper T cells (Tfh cells) were detected using flow cytometry. The functionality of HBV-specific B cells was quantified through ELISpot assays. During treatment, the fraction of activated memory B cells (MBCs) among HBsAg-specific B cells and the expression of IgG, CXCR3, and CD38 increased. The antibody-secretion capacity of HBsAg-specific B cells was only restored in patients achieving a functional cure after treatment and it positively correlated with serum hepatitis B surface antibody levels. The phenotype and function of HBsAg-specific B cells differed between patients with and without functional cure. Patients with functional cure exhibited IgG+ classical MBCs and plasmablasts among HBsAg-specific B cells. HBcAg-specific B cells displayed both attenuated antibody secretion with reduced IgG expression and an IgM+ atypical type of MBC after treatment, irrespective of functional cure. The number of CD40L+ Tfh cells increased after PEG-IFNα treatment and positively correlated with HBsAg-specific B-cell activation. After PEG-IFNα treatment, HBsAg- and HBcAg-specific B cells exhibit various changes in antibody secretion. Their functional differences are reflected in the alterations in phenotypes and subtypes. The presence of CD40L+ Tfh cells is associated with the active recovery of HBsAg-specific B cells. HBV-related complications and hepatocellular carcinoma remain the leading causes of mortality from chronic liver disease worldwide, and a cure is rarely achieved with antiviral therapies. Elucidating the immunological mechanisms underlying the functional cure of patients with chronic hepatitis B offers a promising therapeutic strategy for viral clearance, e.g. via therapeutic vaccination. We analyzed the alterations in HBV-specific B cells in patients treated with pegylated interferon-α and identified novel pathways for immunotherapeutic boosting of B cell immunity.

Live Rotavirus Vaccination Appears Low-risk in Infants Born to Mothers With Inflammatory Bowel Disease on Biologics

Biologic therapies in the context of inflammatory bowel disease and pregnancy lead to improved maternal and fetal outcomes. Placental transfer results in detectable drug concentrations in infants. Rotavirus infection results in diarrheal related hospitalizations; however, the live oral vaccine is not currently recommended in biologic exposed infants. The aim of this study was to assess the effect of maternal biologic therapies on the infant immune system and safety of live rotavirus vaccination in biologic-exposed infants. Biologic-exposed infants underwent standardized clinical assessments, drug concentration, and immune function testing (complete blood count, differential, immunoglobulin levels, extended B-cell and T-cell subset enumeration, Recent Thymic Emigrants, regulatory T-cell numbers, mitogen stimulation assays, and review of T-cell Receptor Excision Circles in the newborn screen). Rotavirus vaccine-specific adverse effects following immunizations up to 42 days post the last dose of the vaccine series were recorded. There were 57 infants born to 52 mothers with inflammatory bowel disease exposed to infliximab (n= 21), adalimumab (n= 19), vedolizumab (n= 10), and ustekinumab (n= 7) in the third trimester for a median of 39 weeks (interquartile range, 38-39 weeks) at delivery. Immunologic assessments validated for age were normal in all infants despite median infliximab concentrations of 6.1 ug/mL (range, 0.4-28.8 ug/mL), adalimumab concentrations of 1.7ug/mL (range, 0.7-7.9 ug/mL), ustekinumab concentrations of 0.6 ug/mL (range, 0-1.1), and undetectable for vedolizumab at 10.7 weeks (interquartile range, 9.4-12.4) of age. The live oral rotavirus vaccine series was provided to 50 infants with the first dose given at a median of 13weeks of age. No adverse effects following immunization were reported. Immune function testing was normal, and administration of live rotavirus vaccination appeared low-risk in biologic-exposed infants irrespective of circulating drug levels.

Interleukin-6 Signaling Blockade Induces Regulatory Plasmablasts in Neuromyelitis Optica Spectrum Disorder.

Interleukin-6 receptor antibodies (IL-6R Abs), including satralizumab, are increasingly used to prevent relapse for neuromyelitis optica spectrum disorder (NMOSD). However, the detailed mechanism of action of this treatment on the lymphocyte phenotype remains unclear. This study focused on B cells in patients with NMOSD, hypothesizing that IL-6R Ab enables B cells to acquire regulatory functions by producing the anti-inflammatory cytokine IL-10. Peripheral blood mononuclear cells were stimulated in vitro to induce the expansion of B-cell subsets, double-negative B cells (DNs; CD19+ IgD-, CD27-) and plasmablasts (PBs; CD19+, CD27hi, CD38hi). Whole B cells, DNs, or PBs were isolated after culture with IL-6R Ab, and IL-10 expression was quantified using quantitative PCR and a cytometric bead array. RNA sequencing was performed to identify the marker of regulatory PBs induced by IL-6R Ab. DNs and PBs were observed to expand in patients with NMSOD during the acute attacks. In the in vitro model, IL-6R Ab increased IL-10 expression in B cells. Notably, IL-10 expression increased in PBs but not in DNs. Using RNA sequencing, CD200 was identified as a marker of regulatory PBs among the differentially expressed upregulated genes. CD200+ PBs produced more IL-10 than CD200- PBs. Furthermore, patients with NMOSD who received satralizumab had a higher proportion of CD200+ PBs than patients during the acute attacks. Treatment with IL-6 signaling blockade elicited a regulatory phenotype in B cells and PBs. CD200+ PBs may be a marker of treatment responsiveness in the context of NMOSD pathophysiology.

B-cell performance in chemotherapy: Unravelling the mystery of B-cell therapeutic potential.

The anti-tumour and tumour-promoting roles of B cells in the tumour microenvironment (TME) have gained considerable attention in recent years. As essential orchestrators of humoral immunity, B cells potentially play a crucial role in anti-tumour therapies. Chemotherapy, a mainstay in cancer treatment, influences the proliferation and function of diverse B-cell subsets and their crosstalk with the TME. Modulating B-cell function by targeting B cells or their associated cells may enhance chemotherapy efficacy, presenting a promising avenue for future targeted therapy investigations. This review explores the intricate interplay between chemotherapy and B cells, underscoring the pivotal role of B cells in chemotherapy treatment. We summarise promising B-cell-related therapeutic targets, illustrating the immense potential of B cells in anti-tumour therapy. Our work lays a theoretical foundation for harnessing B cells in chemotherapy and combination strategies for cancer treatment. Chemotherapy can inhibit B-cell proliferation and alter subset distributions and functions, including factor secretion, receptor signalling, and costimulation. Chemotherapy can modulate complex B-cell-T-cell interactions with variable effects on anti-tumour immunity. Targeting B-cell surface markers or signalling improves chemotherapy responses, blocks immune evasion and inhibits tumour growth. Critical knowledge gaps remain regarding B-cell interactions in TME, B-cell chemoresistance mechanisms, TLS biology, heterogeneity, spatial distributions, chemotherapy drug selection and B-cell targets that future studies should address.

Cutaneous Wound Healing and the Effects of Cannabidiol.

Cutaneous wounds, both acute and chronic, begin with loss of the integrity, and thus barrier function, of the skin. Surgery and trauma produce acute wounds. There are 22 million surgical procedures per year in the United States alone, based on data from the American College of Surgeons, resulting in a prevalence of 6.67%. Acute traumatic wounds requiring repair total 8 million per year, 2.42% or 24.2 per 1000. The cost of wound care is increasing; it approached USD 100 billion for just Medicare in 2018. This burden for wound care will continue to rise with population aging, the increase in metabolic syndrome, and more elective surgeries. To heal a wound, an orchestrated, evolutionarily conserved, and complex series of events involving cellular and molecular agents at the local and systemic levels are necessary. The principal factors of this important function include elements from the neurological, cardiovascular, immune, nutritional, and endocrine systems. The objectives of this review are to provide clinicians engaged in wound care and basic science researchers interested in wound healing with an updated synopsis from recent publications. We also present data from our primary investigations, testing the hypothesis that cannabidiol can alter cutaneous wound healing and documenting their effects in wild type (C57/BL6) and db/db mice (Type 2 Diabetes Mellitus, T2DM). The focus is on the potential roles of the endocannabinoid system, cannabidiol, and the important immune-regulatory wound cytokine IL-33, a member of the IL-1 family, and connective tissue growth factor, CTGF, due to their roles in both normal and abnormal wound healing. We found an initial delay in the rate of wound closure in B6 mice with CBD, but this difference disappeared with time. CBD decreased IL-33 + cells in B6 by 70% while nearly increasing CTGF + cells in db/db mice by two folds from 18.6% to 38.8% (p < 0.05) using a dorsal wound model. We review the current literature on normal and abnormal wound healing, and document effects of CBD in B6 and db/db dorsal cutaneous wounds. CBD may have some beneficial effects in diabetic wounds. We applied 6-mm circular punch to create standard size full-thickness dorsal wounds in B6 and db/db mice. The experimental group received CBD while the control group got only vehicle. The outcome measures were rate of wound closure, wound cells expressing IL-33 and CTGF, and ILC profiles. In B6, the initial rate of wound closure was slower but there was no delay in the time to final closure, and cells expressing IL-33 was significantly reduced. CTGF + cells were higher in db/bd wounds treated with CBD. These data support the potential use of CBD to improve diabetic cutaneous wound healing.

Inhibition of GRK2 ameliorates the pristane-induced mouse SLE model by suppressing plasma cells differentiation

Systemic lupus erythematosus (SLE) is a multifaceted autoimmune disorder characterized by diverse clinical manifestations and organ damage. Despite its elusive etiology, dysregulated subsets and functions of B cells are pivotal in SLE pathogenesis. Peoniflorin-6′-O-benzene sulfonate (CP-25), an esterification modification of Paeoniflorin, exhibits potent anti-inflammatory and immunomodulatory properties in autoimmune diseases (AID). However, the involvement of CP-25 and its target, GRK2, in SLE development has not been explored. In this study, we demonstrate that both genetic deficiency and pharmacological inhibition of GRK2 attenuate autoantibodies production, reduce systemic inflammation, and mitigate histopathological alterations in the spleen and kidney in the pristane-induced mouse SLE model. Importantly, our findings highlight that both genetic deficiency and pharmacological inhibition of GRK2 suppress plasma cells generation and restore dysregulated B-cell subsets by modulating two crucial transcription factors, Blimp1 and IRF4. Collectively, targeting GRK2 with CP-25 emerges as a promising therapeutic approach for SLE.

GFPBW1, a β-glucan from Grifola frondosa as vaccine adjuvant: APCs activation and maturation.

Adjuvants for vaccines with characteristics of improving adaptive immunity particularly via leverage of antigen presenting cells (APCs) are currently lacking. In a previous work we obtained a new soluble 300 kDa homogeneous β-glucan named GFPBW1 from the fruit bodies of Granola frondosa. GFPBW1 could activate macrophages by targeting dendritic cell associated C-type lectin 1 (Dectin-1)/Syk/NF-κB signaling to achieve antitumour effects. In this study the adjuvant effects of GFPBW1 were explored with OVA-antigen and B16-OVA tumor model. We showed that GFPBW1 (5, 50, 500 μg/mL) dose-dependently promoted activation and maturation of APCs in vitro by increasing CD80, CD86 and MHC II expression. We immunized female mice with OVA in combination with GFPBW1 (50 or 300 μg) twice with an interval of two weeks. GFPBW1 markedly and dose-dependently increased OVA-specific antibody titers of different subtypes including IgG1, IgG2a, IgG2b and IgG3, suggesting that it could serve as an adjuvant for both Th1 and Th2 type immune responses. Furthermore, GFPBW1 in combination with aluminum significantly increased the titers of OVA-specific IgG2a and IgG2b, but not those of IgG1, suggesting that GFPBW1 could be used as a co-adjuvant of aluminum to compensate for Th1 deficiency. For mice immunized with OVA plus GFPBW1, no obvious pathological injury was observed in either major organs or injection sites, and no abnormalities were noted for any of the hematological parameters. When GFPBW1 served as an adjuvant in the B16-OVA cancer vaccine models, it could accomplish entire tumor suppression with preventive vaccines, and enhance antitumour efficacy with therapeutic vaccines. Differentially expressed genes were found to be enriched in antigen processing process, specifically increased tumor infiltration of DCs, B1 cells and plasma cells in the OVA plus GFPBW1 group, in accordance with its activation and maturation function of APCs. Collectively, this study systematically describes the properties of GFPBW1 as a novel potent and safe adjuvant and highlights its great potential in vaccine development.

Pharmacodynamic Effect of mTOR Inhibition-based Immunosuppressive Therapy on T- and B-cell Subsets After Renal Transplantation.

The mammalian target of rapamycin inhibitor (mTORi) therapy after kidney transplantation is solely monitored pharmacokinetically, not necessarily reflecting PI3K-Akt-mTOR pathway blockade efficacy leading to potential under-or overimmunosuppression. In this cross-sectional study, phosphoflow cytometry was used to determine the efficacy of mTOR inhibition in peripheral T- and B-lymphocyte subsets by assessing p70S6 kinase (p70S6K) phosphorylation in renal transplant recipients upon treatment with a combination of either mTORi and calcineurin inhibitors (n = 18), or mTORi with mycophenolic acid (n = 9). Nine dialysis patients with end-stage renal disease and 17 healthy age-matched volunteers served as controls. mTORi treatment reduced p70S6K phosphorylation in CD4+, CD8+ T, and CD19+ B cells compared with healthy controls (HCs). Subpopulation analysis of CD4+ T cells and CD19+ B cells revealed a significant reduction of p70S6K phosphorylation in CD4+CD45RA-CD25- Th cells (P < 0.05), CD24hiCD38hi transitional B cells (P < 0.001), CD24+CD38- memory B cells (P < 0.001), and CD24intCD38int-naive B cells (P < 0.05) upon mTORi treatment, whereas CD4+CD45RA-CD25++CD127- regulatory T cells and CD24-CD38hi plasmablasts were not affected. Compared with mTORi + mycophenolic acid therapy, mTORi + calcineurin inhibitor treatment exhibited an even stronger inhibition of p70S6K phosphorylation in CD4+CD45RA-CD25- Th cells and CD8+ T cells. However, trough levels of mTORi did not correlate with p70S6K phosphorylation. mTORi selectively inhibited p70S6K phosphorylation in select lymphocyte subtypes. Assessing p70S6K phosphorylation by phosphoflow cytometry may serve as an approach to understand cell subset specific effects of mTORi providing detailed pharmacodynamic information for individualizing immunosuppression.

Abstract PO-021: Modeling marginal zone lymphomagenesis

Abstract Introduction: Marginal zone lymphomas (MZL) are the second most prevalent subtype of indolent Non-Hodgkin Lymphomas (iNHL), yet lack a unique cytogenetic identifying abnormality. The B-cell receptor signaling pathway is activated in B-cell malignancy and mediates its activity mainly through the Phosphoinositide 3-kinase (PI3K), protein kinase B (Akt), mammalian target of rapamycin (mTOR) pathway. PI3K inhibitors have demonstrated impressive clinical activity in several indolent lymphomas including MZL, underscoring the pivotal role of the PI3K-AKT-mTOR (PAM) pathway in MZL pathogenesis. Furthermore, iNHL overexpress the antiapoptotic protein Bcl-2. Methods: We developed a genetically engineered mouse model with heterozygous global knockout alleles of both the tumor suppressor genes Phosphatase and Tensin Homolog (PTEN) and Liver Kinase B1 (LKB1), resulting in heightened PAM pathway activation across all tissues. We closely monitored these mice for tumor formation via regular physical assessments over several months. Upon tumor detection, mice were euthanized, and tumors were histologically examined. For a more precise representation of B-cell biology akin to the human disease, we employed the Cre-LoxP system to create the CD19-Cre-PTENfl/fl/LKB1fl/fl and CD19-Cre-PTENfl/fl/Eµ-Bcl2. Results: Thirty mice of global KO PTEN+/- LKB1+/- succumbed to or were euthanized due to disease progression, defined as either lymph node enlargement and/or splenomegaly. By Kaplan-Meier analysis, we saw a steady decrease in both tumor-free and overall survival after 3 months of age. Median survival time was 6 months (95% CI: 6, 8). Immunohistochemical analysis of the 51 lymph nodes revealed iNHL in 61.5% (N=32), with all of the MZL subtype. The penetrance rate was 37%. Then, we looked at the absolute B cells, and the major B subtypes in age-matched mice (N=3 per group): WT, CD19-Cre-PTENfl/fl/LKB1fl/fl, CD19-Cre-PTENfl/fl/Eµ-Bcl2. We observed the following preliminary results in the absolute B cells (23,489,812; 1,550,430; 140,476,875), Marginal zone cells (1,087,883; 452,470; 42,658,701), follicular cells (19,078,598; 203,286; 57,212,627), B1 cells (962,192; 939,386; 1,803,060), and plasma cells (211,725; 202,790; 10,531,585) respectively. Then, Kruskal-Wallis tests were used to test the main effect of these three models (WT, CD19-Cre-PTENfl/fl/LKB1fl/fl and CD19-Cre-PTENfl/fl/Bcl2) on absolute cell counts for each cell type: B cells (p=0.027), follicular (p=0.061), Marginal zone (p=0.027), B1 (p=0.43), and plasma cell (p=0.066). The test results indicate that there were statistically discernible differences in absolute cell counts among the three models for B cells and MZ. Conclusion: Marginal zone lymphoma remains an incurable lymphoma with a dearth of reliable preclinical models. Our data provides, for the first time, evidence supporting the involvement of the PAM pathway in MZL pathogenesis, laying the groundwork for further research elucidating the disease biology and rational therapeutic strategies for this incurable malignancy. Citation Format: Emily Harris, Ariel Sindel, Jian He, Roy Sabo, Alden Chesney, Guanhua Lai, Adolpho Mauro, Rebecca Martins, Jennifer Koblinski, Joelene Windle, Victor Yazbeck. Modeling marginal zone lymphomagenesis [abstract]. In: Proceedings of the Fourth AACR International Meeting on Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2024 Jun 19-22; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(3_Suppl):Abstract nr PO-021.

Transcriptome profiling reveals distinct alterations in the B-cell signature and dysregulation of peripheral B-cell subsets in sickle cell anemia patients

Sickle cell anemia (SCA) is characterized by immune system activation and heightened susceptibility to infections. We hypothesized that SCA patients exhibit transcriptional alterations in B-cell-related genes, impacting their peripheral B-cell compartment and leading to dysregulated humoral immunity and increased infection susceptibility. Our objective was to conduct an in silico analysis of whole blood transcriptomes from SCA patients and healthy controls obtained from public repositories. We aimed to identify alterations in the adaptive immune system and validate these findings in our own SCA patient cohort. Bioinformatic analyses unveiled significant transcriptional alterations in B-cell signatures, developmental pathways, and signaling pathways. These results were validated in peripheral blood mononuclear cells from our SCA patient cohort and controls using real-time polymerase chain reaction and flow cytometry. Ninety genes exhibited differential expression, with 70 upregulated and 20 downregulated. Dysregulation in the B-cell compartment of SCA patients was evident, characterized by increased frequencies of immature and naive B-cells, and decreased percentages of memory B-cell subsets compared with healthy controls. Our findings highlight previously unexplored transcriptional and quantitative alterations in peripheral B-cells among SCA patients. Understanding these changes sheds light on the mechanisms contributing to the heightened infection risk in this population. Future studies should delve deeper into these molecular changes to develop targeted interventions and therapeutic strategies aimed at mitigating infection susceptibility in individuals with SCA.

Upregulation of granzyme B and C-X3-C motif receptor 1 in circulating plasmablasts was negatively regulated by Notch signal in patients with systemic lupus erythematosus.

As one molecule related to cytotoxicity, surface expression of C-X3-C motif receptor 1 (CX3CR1) was highly correlated with intracellular granzyme B (GZMB) in NK and cytolytic T cells. However, the expression of CX3CR1 and GZMB in B cells has not been clarified, and their clinical significance in systemic lupus erythematosus (SLE) remains unclear. This study aimed to clarify the changes and clinical significance of peripheral blood B cells expressing GZMB and/or CX3CR1 in SLE. Peripheral blood was collected from 39 SLE patients and 48 healthy controls. We found that GZMB and CX3CR1 expression varied in different B-cell subsets, with plasmablasts possessing the highest positive percentages, consistent with bioinformatics prediction. GZMB+ and CX3CR1+ percentages in circulating B cells and plasmablasts were increased in SLE patients. CX3CR1 was upregulated on B cells after in vitro stimulation. Notch intracellular domain (NICD) expression was significantly decreased in plasmablasts of SLE patients and CX3CR1 in plasmablasts was downregulated with the addition of JAG1. In conclusion, GZMB and CX3CR1 were increased in B cells and in plasmablasts of SLE patients and CX3CR1 was negatively regulated by Notch signal in plasmablasts, which may be involved in SLE pathogenesis.

A Case of Primary Thyroid Maltoma

Mucosa-associated lymphoid tissue (MALT) lymphomas comprise 7.6% of non-Hodgkin’s lymphomas (NHLs) and they are recently recognized B-cell subset of NHLs. They are originated from gastrointestinal tract most frequently but may also occur in other organs including head and neck, lung, skin, thyroid and breast. Primary thyroid lymphomas (PTLs) constitute up to 5% of all thyroid malignancies. Diffuse large B-cell lymphoma is most common type of PTLs and MALT lymphoma is relatively rare subtype of PTLs. Thyroid MALToma arises in chronic inflammatory conditions with autoimmune or infectious etiologies. The optimal treatment regimen still remains controversial. However, It is reported that localized thyroid MALT lymphoma has excellent survival rate after surgical resection alone. We report a case of 48-years-old woman with primary thyroid MALToma.

Outcomes of Human Leukocyte Antigen-Matched Related Donor and Haploidentical Allogeneic Hematopoietic Cell Transplantation Recipients by Immune Profiles of Recipients and Donors

Haploidentical (Haplo) allogeneic HCTs (alloHCT) have been used more frequently over the last decade as survival is similar to HLA-matched related donor (MRD) alloHCTs. We aimed to identify donor and recipient immune signatures before alloHCT that are associated with clinically meaningful outcomes in MRD vs Haplo alloHCT recipients. This retrospective cohort study of 165 MRD (n = 132) and Haplo (n = 33) alloHCT recipients and their related donors between 2007-2019 with paired peripheral blood samples immunophenotyped for T-cell, B-cell, NK cell and dendritic cell (DC) subsets. Immune cells were quantified before alloHCT in donors and recipients; calculations of immune cell ratios were classified as high, intermediate, and low and analyzed with alloHCT outcomes. Haplo donors were younger than MRD donors (median: 35 vs 51 years), whereas Haplo recipients were older than MRD recipients (median: 68 vs 54 years), were more likely to have a Karnofsky Performance Score ≤ 70 (76% vs 57%), 3+ comorbidities (54% vs 47%), and were in complete remission prior to alloHCT (58% vs 42%). In MRD alloHCT, a lower ratio of CD4+ to CD8+ effector memory cells in the donor was associated with lower 4-yr overall survival (OS; 25% vs 61%; P = .009), lower 4-yr progression free survival (PFS; 25% vs 58%; P = .014) and higher incidence of 1-yr transplant-related mortality (TRM; 39% vs 7%; P = .009) in recipients. A higher ratio of CD8+ effector memory to total NK cells measured in MRD recipients was associated with a higher incidence of grade II-IV aGvHD (63% vs 37%; P = .004) but was not statistically significant for III-IV aGvHD (23% vs 12%). In Haplo alloHCT, a lower ratio of total T-regulatory to CD4+ central memory cells in the donor was associated with lower 4-yr PFS (22% vs 60%; P = .0091). A higher ratio of CD4+ effector memory to CD8+ effector memory cells measured in Haplo recipients pre-alloHCT was associated with lower 4-yr OS (25% vs 88%; P = .0039). In both MRD and Haplo recipients, a higher ratio of CD4+ naïve to CD4+ central memory cells was associated with a higher incidence of grade II-IV aGvHD (64% vs 38%; P = .04). Evaluation of pre-alloHCT immune signatures of the donor and recipient may influence clinically meaningful patient outcomes in both MRD and Haplo transplants.

#953 Preliminary results of the peripheral T follicular cells and their subsets in ANCA associated glomerulonephritis

Abstract Background and Aims T cells play crucial role in the pathogenesis of autoimmune diseases such as ANCA associated vasculitis-glomerulonephritis (AAV/GN) by secreting immune mediators and helping B cell-mediated long-lived humoral immunity development. Specific T and B cells subsets can be used as biomarkers of patients with AAV/GN. We aimed to prospectively compare the predictive ability of such subpopulations in patients with active AAV/GN. Method Flow cytometry was applied in the peripheral blood of 29 subjects. 15 AAV/GN patients (M/F 6/9), age: 62.1(28-82)years, MPO/PR3/Negative ANCA: 10/4/1) and 14 healthy controls (HC), to estimate T-cells subsets: CD4, T-folicular cells (Tfol), T-regulatory cells (Tregs), Tfh, Tfh1, Tfh2, Tfh17, and T-folicular regulatory cells(Tfr) and B-cells subsets: CD19, IgD(+)CD127(-), IgD(+)CD127(+), IgD(-)CD127(+) and IgD(-)CD127(-). Results Compared to HC, AAV/GN patients had reduced CD4 (698.8 Vs 368.8, p = 0.04) but increased proportion of Tfol, and both Tfh1 and Tfh2 cells (7% vs 12%, p = 0.05, 8.4 vs 11.2, p = 0.03, 13.3 vs. 45.9, p = 0.09, respectively). Both Tfr (p = 0.048) and Tfh (p = 0.023) were also significantly higher in group A, compared to group HC. CD19 levels were lower in AAV/GN compared to HC, with reduced all subpopulation, though of not-statistical significance. Conclusion Specific T-cells subsets can be measured in AAV/GN patients’ peripheral blood. Subpopulations such as Tfh and Tfr can be used as biomarkers to diagnose patients with active disease and their role in the pathogenesis of AAV/GN must be studied in detail to determine if they can become targets for new therapies in the future.