IgM Expression Research Articles

Unravelling the ontogeny and tissue-specific expression profiles of immune-related genes in the near-threatened endemic catfish, Clarias dussumieri

Clarias dussumieri, an air breathing catfish endemic to the Western Ghats in India, is categorized as 'Near Threatened' by the IUCN. This species is of high regional consumer demand and is one of the prioritized candidate species for aquaculture diversification and conservation. Despite its ecological and commercial significance, comprehensive studies on its immune system are lacking. This study elucidates the ontogenetic development and tissue-specific (brain, anterior kidney, gill, spleen, muscle, liver, hindgut and skin) expression profiles of key immune-related genes in C. dussumieri. Larvae were sampled at various developmental stages post-fertilization, and tissues from adult fish were analyzed for expression patterns of genes associated with inflammation (IL-1β, TNF-α, iNOS), antimicrobial defense (LYS, HAMP), stress response (HSP70), complement system (C3), cell-mediated immunity (MHC-IIβ, CD4-1), and adaptive immunity (IgM). The expression of IL-1β was highest at 60 days post-fertilization (60D), while TNF-α expression peaked at 25D before dropping notably by 60D. iNOS showed a peak at 7D, underscoring its importance in early immune defence. LYS exhibited a high expression at 10D, while HAMP peaked at 60D, highlighting their roles in antimicrobial defence. Stress marker HSP70 increased from 15D onwards and complement component C3 was consistently expressed at low levels throughout development. MHC-IIβ and CD4-1 showed significant increase since 10D and 7D respectively, suggesting the establishment of cell-mediated immunity. IgM expression increased notably from 15 days, indicating the development of adaptive immunity. In adult fish tissues, IL-1β, TNF-α, LYS and HSP70 showed highest expression in the hindgut, while C3 was predominantly expressed in the liver. MHC-IIβ and CD4-1 were highly expressed in the spleen and anterior kidney respectively. IgM was abundant in the anterior kidney and spleen. This study provides crucial baseline data on the immune competence of different developmental stages of C. dussumieri, informing strategies for effective vaccination and disease management in aquaculture, and enhancing our understanding of fish immunology for conservation efforts.

Immunological resilience of a temperate catshark to a simulated marine heatwave.

Marine heatwaves (MHWs) have recently been proposed to be more relevant in driving population changes than the continuous increase in average temperatures associated with climate change. The causal processes underpinning MHW effects in sharks are unclear but may be linked to changes in fitness caused by physiological trade-offs that influence the immune response. Considering the scarcity of data about the immune response of sharks under anomalous warming events, the present study analyzed several fitness indices and characterized the immune response (in the blood, epigonal organ, liver, spleen and intestine) of temperate adult small-spotted catsharks (Scyliorhinus canicula) after a 30 day exposure to a category II MHW. The results indicated that adult small-spotted catsharks have developed coping strategies for MHWs. Specifically, among the 35 parameters investigated, only the gonad-to-body ratio (GBR) and plasma glucose concentration showed significant increases. In contrast, gene expression of igm and tumor necrosis factor receptor (tnfr) in blood cells, and tnfr in the epigonal organ, as well as the number of monocytes, all significantly decreased. Although a decline in immune function in small-spotted catsharks was revealed following MHW exposure, energy mobilization restored homeostasis and indicated a shift in energy allocation towards reproduction. Group resilience may be due to the variable tolerance of individuals, the phenotypic plasticity of cellular immunity, thermal imprinting and/or metabolic capacity of the individuals.

Isolation and identification of hybrid snakehead rhabdovirus (HSHRV) and its immune response in the hybrid Snakehead((male Channa argus × female Channa maculata)

Hybrid snakehead is an emerging aquaculture species obtained from the mating of Channa argus (♂) and Channa maculate (♀). It has the advantages of fast growth and strong disease resistance. Viral diseases caused by hybrid snakehead rhabdovirus (HSHRV) critically affect the hybrid snakehead industry. We isolated and identified a highly virulent strain of HSHRV from a naturally occurring hybrid snakehead, namely HSHRV-GZ22. It showed clinical signs of sinking, superficial blackening, spinning, acute internal congestion, and hemorrhage, along with blackening and enlargement of the liver, spleen, and kidneys. Histopathological analysis showed multiple tissue lesions in the liver, spleen, and kidneys, characterized mainly by massive inflammatory cell infiltration, interstitial hemorrhage, and partial cell necrosis. Pathogen analysis identified the virus as HSHRV. Immunofluorescence analysis (IFA) with HSHRV-specific antibodies confirmed the virus and electron microscopic observation showed that the bullet-like virus particles had a size of approximately 150 nm. The replication efficiency of HSHRV was 107.33 TCID50/mL. The glycoproteins of the isolates were cloned and sequenced, and a phylogenetic tree was constructed. The HSHRV-GZ22 isolates clustered into a single branch with the reported HSHRV-C1207, and it had a high degree of homology with Siniperca chuatsi rhabdovirus (SCRV). HSHRV-GZ22 was regressively infected, clinical and pathological symptoms were similar to naturally occurring fish, with a fatality rate of about 85 %. qRT-PCR was performed to determine the viral replication in different tissues of hybrid snakehead, and the viral copies were found to be highly expressed in the liver, spleen, kidney, and intestine. HSHRV-GZ22 activated the antiviral immune pathway in hybrid snakeheads during infection, and the expressions of IgM, IRF7, ISG12, and IFNγ were significantly altered. In this study, we isolated a strong virulent strain of HSHRV and characterized it; in addition, it provided insights into the pathogenesis of HSHRV and immune response in hybrid snakehead, while also advancing the methods for diagnosing and preventing diseases caused by HSHRV.

Vaccinated Atlantic salmon (Salmo salar L.) maintain a specific antibody response throughout the seasonal fluctuations of a full commercial production cycle in sea: a case study

Vaccination is a critical prophylactic measure to prevent disease outbreaks in Atlantic salmon (Salmo salar L.) and multivalent oil-adjuvanted vaccines administered intraperitoneally (IP) are widely used for this purpose. The Atlantic salmon in this observational study were sampled during commercial production in a salmon farm in Northern Norway. Fish were sampled in the hatchery prior to vaccination, and further every 6 weeks after sea transfer up until slaughter. The key findings revealed that Atlantic salmon vaccinated IP with a commercial multivalent oil-based vaccine developed specific and long-lasting antibody responses against A. salmonicida, V. anguillarum, Y. ruckeri, and IPNV for up to 20 months post-vaccination. However, there was variation in the measured specific antibody response to the different antigens. Notably, the antibody responses against A. salmonicida and V. anguillarum antigens were higher and more prolonged compared to the antibody responses against Y. ruckeri and IPNV antigens. Histological examination showed presence of vaccine-induced granulomas in the pancreas at the injection site for up to 50 weeks post-vaccination and V. anguillarum antigens were found in the granulomas up to 44 weeks post-vaccination. There were also individual IgM-positive cells present in these vaccine-induced granulomas, and qPCR confirmed increased gene expression of membrane-bound and secretory IgM up to 37 weeks post-vaccination. Altogether the qPCR data of membrane and secretory IgM suggests local production of IgM in the vaccine-induced granulomas, while the head kidney might be responsible for the long-term secretion of IgM. Neither the low temperature the fish experienced for two winter seasons (below 5 °C) nor the seasonal changes in photoperiod affected the vaccine-induced specific antibody responses.

Effect of Leishmania infantum infection on B cell lymphopoiesis and memory in the bone marrow and spleen.

Visceral leishmaniasis (VL) is characterized by an uncontrolled infection of internal organs such as the spleen, liver and bone marrow (BM) and can be lethal when left untreated. No effective vaccination is currently available for humans. The importance of B cells in infection and VL protective immunity has been controversial, with both detrimental and protective effects described. VL infection was found in this study to increase not only all analyzed B cell subsets in the spleen but also the B cell progenitors in the BM. The enhanced B lymphopoiesis aligns with the clinical manifestation of polyclonal hypergammaglobulinemia and the occurrence of autoantibodies. In line with earlier reports, flow cytometric and microscopic examination identified parasite attachment to B cells of the BM and spleen without internalization, and transformation of promastigotes into amastigote morphotypes. The interaction appears independent of IgM expression and is associated with an increased detection of activated lysosomes. Furthermore, the extracellularly attached amastigotes could be efficiently transferred to infect macrophages. The observed interaction underscores the potentially crucial role of B cells during VL infection. Additionally, using immunization against a fluorescent heterologous antigen, it was shown that the infection does not impair immune memory, which is reassuring for vaccination campaigns in VL endemic areas.

IgT-mediated mucosal immunity and microbiota dynamics in snakehead (Channa argus) post Aeromonas veronii TH0426 and Aeromonas hydrophila TPS infection: implications for aquaculture disease management.

The aquaculture sector, vital to global food security, grapples with bacterial pathogens compromising fish health and industry sustainability. This investigation probes mucosal immune responses and gut microbiota dynamics in snakehead (Channa argus) post-Aeromonas infection, a prevalent aquaculture challenge. Employing infection models, we delineated the integral role of immunoglobulin T (IgT) in mucosal immunity and its interaction with gut microbiota. Fish from a local farm, maintained under controlled conditions, were infected with Aeromonas veronii TH0426 and Aeromonas hydrophila TPS. Post-infection, daily monitoring and sample collection at specified intervals were conducted for comprehensive analysis. Histopathology, quantitative PCR, immunofluorescence, and microbiota profiling revealed significant immune and microbial changes, particularly at day 7. Intestinal IgT, IgM, and pIgR gene expression surged, indicative of a robust response. Immunofluorescence microscopy confirmed increased IgT+ and pIgR+ cell infiltration in the epithelium. Post-infection dysbiosis, with altered bacterial composition, was partially offset by elevated IgT levels. These insights underscore IgT's crucial function in mucosal defense and suggest potential for probiotic and vaccine strategies to enhance aquaculture disease resilience.

Interleukin-1 beta (IL-1β) as adjuvant enhances the immune effects of Aeromonas veronii inactivated vaccine in largemouth bass (Micropterus salmoides)

Largemouth bass (Micropterus salmoides) has emerged as a significant economic fish species, with a rise in Aeromonas veronii infections in farming. However, research on adjuvants for vaccines against A. veronii in largemouth bass remains scarce. In present study, recombinant largemouth bass IL-1β (LbIL-1β) was expressed to explore its adjuvant effect on the A. veronii inactivated vaccine. Following vaccination with recombinant LbIL-1β (rLbIL-1β) and the inactivated A. veronii, higher serum SOD levels and lysozyme activities were observed in largemouth bass from inactivated A. veronii + rLbIL-1β vaccinated group. Furthermore, it was discovered that rLbIL-1β was able to boost the serum-specific antibody levels induced by the inactivated A. veronii. The qRT-PCR analysis revealed that rLbIL-1β also enhanced the expression of IgM, CD4, and MHC II in largemouth bass triggered by the inactivated A. veronii. After challenged with live A. veronii, the outcomes demonstrated that the relative percentage survival (RPS) for largemouth bass resulting from the inactivated A. veronii in combination with rLbIL-1β was 76.67 %, surpassing the RPS of 60 % in the inactivated A. veronii group. Collectively, these findings indicate that rLbIL-1β enhances the protective effect of the A. veronii inactivated vaccine on largemouth bass, showcasing potential as an adjuvant for further development.

Identification and Functional Analysis of a de novo IKZF3 Mutation in a Pediatric Patient with Combined Immunodeficiency.

AIOLOS, a vital member of the IKAROS protein family, plays a significant role in lymphocyte development and function through DNA binding and protein-protein interactions. Mutations in the IKZF3 gene, which encodes AIOLOS, lead to a rare combined immunodeficiency often linked with infections and malignancy. In this study, we evaluated a 1-year-4-month-old female patient presenting with recurrent infections, diarrhea, and failure to thrive. Laboratory investigations revealed decreased T lymphocyte and immunoglobulin levels. Through whole-exome and Sanger sequencing, we discovered a de novo mutation in IKZF3 (NM_012481; exon 5 c.571G > C, p.Gly191Arg), corresponding to the third DNA-binding zinc finger region of the encoded protein AIOLOS. Notably, the patient with the AIOLOS G191R mutation showed reduced recent thymic emigrants in naïve CD4+T cells compared to healthy counterparts of the same age, while maintaining normal levels of Th1, Th2, Th17, Treg, and Tfh cells. This mutation also resulted in decreased switched memory B cells and lower CD23 and IgM expression. In vitro studies revealed that AIOLOS G191R does not impact the expression of AIOLOS but compromises its stability, DNA binding and pericentromeric targeting. Furthermore, AIOLOS G191R demonstrated a dominant-negative effect over the wild-type protein. This case represents the first reported instance of a mutation in the third DNA-binding zinc finger region of AIOLOS highlighting its pivotal role in immune cell functionality.

Functional role of TrIL-1β in Takifugu rubripes defense against Cryptocaryon irritans infection

The Japanese puffer, Takifugu rubripes, is a commercially important fish species in China that is under serious threat from white spot disease (cyptocaryoniasis), which leads to heavy economic losses. We previously found that interleukin-1β (IL-1β), an important cytokine with a potential role in resistance against pathogens, was one of the most significantly differentially up-regulated proteins in the gills and spleen of T. rubripes infected by the protozoan parasite Cryptocaryon irritans. In this study, we assessed the potential function of T. rubripes IL-1β (TrIL-1β) in fish infected with C. irritans. Phylogenetic analysis indicated that the TrIL-1β protein sequence was most closely related to that of Atlantic salmon (Salmo salar) (67.2 %). The incubation experiments revealed that TrIL-1β may reduce trophont activity by destroying membranes. Immunofluorescence experiments showed that recombinant TrIL-1β promoted the expression of endogenous IL-1β, which penetrated and disrupted the cell membranes of trophonts. Transmission electron microscopy showed that the IL-1β group had less tissue damage compared with control groups of fish. IL-1β-small interfering RNA and IL-1β overexpression experiments were performed in head kidney primary cells, and challenge experiments were performed in vitro. Quantitative RT-PCR results showed that TrIL-1β regulated and activated MyD88/NF-κB and MyD88/MAPK/p38 signaling pathways during C. irritans infection. TrIL-1β also promoted the differential expression of IgM, showing that it was involved in humoral immunity of T. rubripes. The cumulative mortality experiment show that TrIL-1β could protect fish against C. irritans infection. These results enrich current knowledge about the molecular structure of TrIL-1β. They also suggested that recombinant TrIL-1β could be used as an adjuvant in a subunit vaccine against C. irritans infection, which is of profound importance for the prevention and control of parasitic diseases in T. rubripes.

Presence of CoV-2 antibody in vitreous humor after Cov-2 infection

PurposeCoronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a life-threatening disease with largely unknown intraocular pathogenesis. Herein, we determined the presence of SARS-CoV-2-specific ribonucleic acid (RNA) and virus-associated antibodies in the vitreous humor of people who have recently recovered from SARS-CoV-2 infection. DesignThis cross-sectional study included 33 patients (33 eyes) who have recently recovered from SARS-CoV-2 infection. Vitreous humor and blood serum samples were tested for the SARS-CoV-2 RNA and virus-associated antibodies. ResultsAmong 33 participants, blood serum and vitreous humor were all tested negative for SARS-CoV-2 RNA. SARS-CoV-2-specific IgM was detected in 87.88 % (29/33) patients in blood serum and 6.10 % (2/33) in vitreous humor; SARS-CoV-2-specific IgG was detected in 96.97 % (32/33) patient in blood serum and 81.82 % (27/33) in vitreous humor. Statistical significance was found for IgM expression between blood serum and vitreous humor (P < 0.01), while IgG was not (P = 0.11). The days after recovery were statistically longer both in IgM-positive blood serum samples group and IgG-positive vitreous humor samples group compared with negative samples of each group (P < 0.01). Additionally, no statistical difference could be detected in antibody expression in vitreous humor between different groups divided on the condition of the risk of blood-retina-barrier (BRB) failure (P = 0.49 for IgM; P = 0.37 for IgG). ConclusionAfter recovering from COVID-19, no SARS-CoV-2 RNA was detected in vitreous humor, but anti-CoV-2 IgM was detected in 6.1 % and IgG in approximately 80 % of vitreous humor samples of participants. We also found that the positivety rate of SARS-CoV-2-specific antibodies in the blood serum and vitreous humor were both correlated with the days after recovery since the infection.

Trans-Cinnamaldehyde Primes More Robust Channel Catfish Immune Responses to Edwardsiella ictaluri Infection.

Infection with Edwardsiella ictaluri, a causative agent of enteric septicemia of catfish, threatens profitable catfish production through inventory losses. We previously demonstrated that trans-cinnamaldehyde (TC) enhances the survival of catfish following E. ictaluri infection. The present study was conducted to investigate catfish immune responses to TC feeding and E. ictaluri infection. The expression of 13 proinflammatory, innate, and adaptive immune-related genes was evaluated over time in two sets of experiments using real-time polymerase chain reaction (PCR). In the first experiment, catfish were fed a basal diet with or without TC supplementation, while in the second they were fed a TC-supplemented or normal diet followed by infection with E. ictaluri. The catfish group infected with E. ictaluri and fed a TC-diet showed significant changes in the expression of innate and adaptive immune-related genes compared to control group. At 21 and 28 days post-infection, recovered fish showed significant increases in the expression of IgM in the anterior kidney and spleen. These results suggest that the supplemental dietary intake of TC can improve the immune status of catfish via engaging innate and adaptive immune responses and the production of memory cells in immunocompetent tissues. Together, this study provides an important foundation for the potential application of TC as an antimicrobial alternative in aquaculture.

Antigen-independent, autonomous B cell receptor signaling drives activated B cell DLBCL.

Diffuse large B cell lymphoma of activated B cell type (ABC-DLBCL), a major cell-of-origin DLBCL subtype, is characterized by chronic active B cell receptor (BCR) signaling and NF-κB activation, which can be explained by activating mutations of the BCR signaling cascade in a minority of cases. We demonstrate that autonomous BCR signaling, akin to its essential pathogenetic role in chronic lymphocytic leukemia (CLL), can explain chronic active BCR signaling in ABC-DLBCL. 13 of 18 tested DLBCL-derived BCR, including 12 cases selected for expression of IgM, induced spontaneous calcium flux and increased phosphorylation of the BCR signaling cascade in murine triple knockout pre-B cells without antigenic stimulation or external BCR crosslinking. Autonomous BCR signaling was associated with IgM isotype, dependent on somatic BCR mutations and individual HCDR3 sequences, and largely restricted to non-GCB DLBCL. Autonomous BCR signaling represents a novel immunological oncogenic driver mechanism in DLBCL originating from individual BCR sequences and adds a new dimension to currently proposed genetics- and transcriptomics-based DLBCL classifications.

Investigation of IL-6 serum level in COVID-19 patients with positive COVID-19 IgG/IgM antibody titers to check inflammation and disease progression

ObjectiveThe SARS-CoV-2, the cause of coronavirus disease 2019 (COVID-19), leads to severe pathogenicity and high mortality among different communities around the world. Therefore, it is important to understand the mechanisms of virus pathogenesis and the immune system's response to prevent the further spread of this virus. This study was aimed to evaluate the relationship between the serum level of interleukin 6 and positive IgG and IgM antibody levels in patients with COVID-19 to investigate inflammation and disease progression. Methods & MaterialsIn this study, 10 ml of EDTA blood samples were taken from 414 COVID-19 patients. Then, the plasma was separated and the levels of IgM and IgG antibodies and interleukin 6 cytokine were evaluated by ELISA and chemiluminescence methods, respectively. All data were analyzed by SPSS 22 and GraphPad prism 9 software at the significance level of P < 0.05. ResultsThe results of this study showed that there was no significant difference in the expression of IgM and IgG antibodies between men and women. Also, a significant increase in the mean expression of IL-6 was observed only in the high concentration range (100−〉1000 pg/ml) in men compared to women (P < 0.001). In addition, in the female population, all three concentration ranges (negative, medium, and high) of IL-6 have the highest correlation with high titers (>10 U/ml) of IgM and IgG antibodies. While, in men, all three concentration ranges of IL-6 had the highest correlation with > 10 U/ml IgM antibody titers, but in the case of IgG, the highest correlation between different concentrations of IL-6 was observed with the negative or moderate titers of this antibody and there was an inverse relationship with the high titers of IgG (>10 U/ml). ConclusionAs a result, the relationship between different serum levels of cytokine IL-6 with different titers of IgM and IgG antibodies was observed in both male and female populations. In general, it can be concluded that the correlation between different concentrations of IL-6 with different IgM titers was similar in both men and women, but in the case of different IgG titers, this correlation was higher in women than men.

Contrasting roles for IgM and B-cell MHCII expression in Brucella abortus S19 vaccine-mediated efficacy against B. melitensis infection.

Brucella is a neglected zoonotic pathogen with a worldwide distribution. Our study delves into B-cell effector functions in live vaccine-mediated immunity against brucellosis. Notably, we found antibody production, particularly secretory IgM, confers protection against virulent Brucella melitensis in vaccinated mice, which was associated with complement activation. By contrast, B-cell MHCII expression negatively impacted vaccine efficacy. In addition, B-cell depletion after vaccination, but before the B. melitensis challenge, enhanced protection against infection, suggesting a detrimental B-cell role during the challenge phase. Interestingly, deficiency of T follicular helper cells, which are crucial for aiding germinal center B cells, dampened vaccine efficacy at later stages of challenge independent of antibody production. This study underscores contrasting and phase-dependent roles of B-cell effector functions in vaccine-mediated immunity against Brucella.

The Efficacy of Nile Tilapia Vaccination by Formalin killed Streptococcus agalactiae Encapsulated in Alginate Microcapsules

Nile tilapia is one of the major farmed fish that is often infected with Streptococcus. Oral vaccination is the most preferable technique, but the antigen is usually degraded by gastric juice. Encapsulation by alginate could be effective in protecting the antigen. The aim of this study was to assess the effectiveness of vaccinating Nile tilapia using formalin-killed Streptococcus agalactiae encapsulated in alginate. The average diameter of fabricated microcapsules was 500±20 μm, with 86% encapsulation efficacy. Fish were then divided into four groups (30 fish per group): group A: fish vaccinated by alginate microcapsules covered by formalin-killed cells (FKC), group B: fish vaccinated by non-covered FKC, group C: fish fed with empty alginate microcapsules and group D: fish feed with commercial pellets as control group. All groups were treated for 14 days and then fed with commercial pellets. Blood samples were collected on days 7, 14, 21, and 28 days post-vaccination. Bactericidal activity, lysozyme activity and serum antibody titer were significantly elevated in group A (p&lt;0.001) compared to other treated groups. Then, gene expression analysis was performed at 29 days post-vaccination to evaluate the expression of main immune contributors such IgM, IgT, TCR β, CD4, CD8α, IL 1β, IL 8, IFN 1, and TNFα. The analysis showed significant upregulation of all tested genes in group A compared to other treated groups (p&lt;0.001). Lastly, a challenge test was done on day 30 post-vaccination by injecting 4.6×106 CFU mL−1 of virulent S. agalactiae. The relative percent of survival (RPS) were 92± 2%, 48± 5% and 4± 3% for groups A, B and C respectively. The obtained result indicated that alginate encapsulation provided antigen protection due to its higher immunogenicity compared to non-covered vaccine. Hence, the fabricated vaccine could be incorporated with food and orally administered to Nile tilapia to prevent S. agalactiae infection.

Codon optimization regulates IgG3 and IgM expression and glycosylation in N. benthamiana.

Plants are being increasingly recognized for the production of complex human proteins, including monoclonal antibodies (mAbs). Various methods have been applied to boost recombinant expression, with DNA codon usage being an important approach. Here, we transiently expressed three complex human mAbs in Nicotiana benthamiana, namely one IgG3 and two IgM directed against SARS-CoV-2 as codon optimized(CO) and non-codon optimized (NCO) variants. qRT-PCR exhibited significantly increased mRNA levels of all CO variants compared to the non-codon optimized orthologues, in line with increased protein expression. Purified CO and NCO mAbs did not exhibit obvious biochemical differences, as determined by SDS-PAGE and antigen binding activities. By contrast, enhanced production selectively impacts on glycosite occupancy and N-glycan processing, with increased mannosidic structures. The results point to a careful monitoring of recombinant proteins upon enhancing expression. Especially if it comes to therapeutic application even subtle modifications might alter product efficacy or increase immunogenicity.

Effects of Oregano Essential Oil on IgA+, IgG+, and IgM+ Cells in the Jejunum of Castrated Holstein Bulls.

The aim of this study was to investigate the effect of oregano essential oil on IgA+, IgG+, and IgM+ cells in the jejunum of castrated Holstein bulls. Twelve castrated Holstein bulls were randomly divided into control (YCK) and oregano essential oil (YEO) groups. Pathological changes in the jejunum were observed by HE staining, and the expression levels of IgA, IgG, and IgM in the jejunum were detected by ELISA. The distributions of IgA+, IgG+, and IgM+ cells in the jejunum were analysed by multiplex immunofluorescence and immunohistochemistry. The results showed that the jejunal villi were detached in the YCK group, which may have been related to inflammation, while the intestinal epithelium was clear and intact in the YEO group. The expressions of IgA, IgG, and IgM were significantly reduced by 40.75%, 30.76%, and 50.87%. The IgA+, IgG+, and IgM+ cells were diffusely distributed in the lamina propria of the jejunum, and were reduced by 17.07%, 6.44%, and 6.15%, respectively. Oregano essential oil did not alter the distribution characteristics of IgA+, IgG+, or IgM+ cells in the jejunum, but it suppressed inflammatory response, decreased immunoglobulin content, and significantly enhanced the formation of an immune barrier in the gastrointestinal mucosa.

CD49d Shapes B Cell Receptor Responsiveness and Tissue Distribution in an Aggressive Chronic Lymphocytic Leukemia Murine Model

Background: High expression of the very late antigen-4 (VLA-4) integrin subunit CD49d predicts disease progression in chronic lymphocytic leukemia (CLL). We previously demonstrated that B cell receptor (BCR) signals induce activation of VLA-4 (Tissino et al., J Exp Med. 2018) and characterized Eµ-TCL1-transgenic (TCL1-tg) mice as an adequate model for the human high-risk CD49d-high CLL patient subgroup, with VLA-4 inhibition hampering the disease (Szenes et al., Leukemia 2020). Here, we address the consequences of genetic B cell-specific ablation of CD49d on disease development, engraftment capacity and treatment susceptibility in a novel CLL mouse model. Methods: We crossed ITGA4fl/fl mice, kindly provided by T. Papayannopoulou, University of Washington, with TCL1-tg mice and CD19 Cre mice, subsequently named CD49d∆B TCL1-tg. Tumor development was continuously monitored by regular blood checks and mice were sacrificed at 10 months. Tumor infiltration was quantified in peritoneal cavity (PC), spleen and bone marrow by flow cytometry using anti-CD5/CD19 antibodies. Integrin and chemokine receptor expression was profiled. In vitro responsiveness to BCR stimulation of CD49d-deficient and proficient leukemic cells was assessed using phospho-specific flow cytometry. Furthermore, purified leukemic cells derived from PC or spleen were subjected to RNA-seq analyses. CD49d-deficient or proficient leukemic cells were transplanted into wild-type C57BL/6J recipients in short and long term experiments. Ongoing: CD49d-deficient or proficient leukemic cells were transplanted competitively into C57BL/6J recipients and are receiving continuous Bruton's tyrosine kinase (BTK)-inhibitor (acalabrutinib) treatment upon tumor onset. Results: CD49d∆B TCL1-tg mice showed a B cell-specific integrin loss in all organs. Blood tumor burden was measured monthly. At months seven and eight, leukemic cells of CD49d∆B TCL1-tg mice were reduced by approx. 25% compared to TCL1-tg littermates (Fig. 1A). This trend was reversed at month nine, with approx. 45% increased lymphocytosis in CD49d∆B TCL1-tg mice. CD49d expression in leukemic cells of TCL1-tg mice was unaffected by disease progression. At sacrifice, CD49d∆B TCL1-tg mice showed an 80% ( p=&amp;lt;0.0001) reduction of tumor burden in the bone marrow (Fig. 1B), but comparable leukemic cell numbers in PC and spleen. Leukemic splenocytes derived from CD49d∆B TCL1-tg mice displayed 38% ( p=0.032) higher IgM and 33% higher CD44 expression but 43% ( p=0.015) reduced expression of CXCR4. BCR signaling was modulated by the integrin loss and BCR stimulation with anti-IgM induced 43% ( p=0.022) higher PLCγ2 and 45% ( p=0.022) higher SYK phosphorylation in leukemic cells derived from CD49d∆B TCL1-tg mice compared to littermates, but comparable BTK phosphorylation. RNA-seq analysis revealed strong differences in leukemic cell transcriptomes between PC and spleen, indicating tissue dependent effects on leukemic cells. Particularly, TNF-α signaling via NF-κB, MTORC1, MAPK signaling and JAK-STAT signaling pathways were upregulated in leukemic cells derived from spleen compared to PC irrespective of CD49d expression, while chemokine signaling pathways were downregulated in PC. When comparing CD49d deficient and proficient leukemic cells, the gene set MYC targets v1 was enriched in deficient cells. When injecting CD49d-deficient leukemic splenocytes intravenously, more cells were recovered after 5 days from blood ( p=0.048) and spleen compared to CD49d-proficient cells, but did not engraft in bone marrow or peritoneum. When injected intraperitonally, CD49d-deficient leukemic cells were found in peritoneum in contrast to TCL1-tg littermates ( p=&amp;lt;0.0001). Conclusion: The TCL1 mouse model resembles a human high-risk CLL (e.g. IGHV unmutated, CD49d-expressing), and thus allows to investigate the impact of CD49d expression in this CLL subset. Loss of CD49d expression resulted in an altered redistribution of leukemic cells in tissue sites with a significant reduction of leukemic cells in the bone marrow. Lack of integrin signals resulted in increased antigen-induced BCR signaling and IgM expression, as well as a B cell receptor activation phenotype associated with MYC gene set enrichment, indicating a feedback loop from integrins to the BCR. Currently, the susceptibility of CD49d∆B TCL1-tg mice to BTK inhibitors is under investigation.

Precision Targeting of the Malignant Clone in Diffuse Large B Cell Lymphoma Using Chimeric Antigen Receptor T Cells Against the Clonotypic IGHV4-34 B Cell Receptor

Introduction: CD19-directed Chimeric Antigen Receptor T cell (CART19) immunotherapy has revolutionized the treatment of B cell lymphoma. However, most CART19-treated patients either fail to respond or show disease progression after an initial response. In fact, up to 30% of lymphoma relapses following CART19 show loss of CD19 expression. In addition, several patients who respond well to CART19 display complete loss or severe reduction of their normal B cell repertoire, leaving them exposed to recurrent infections, limited response to vaccines, and requiring prophylactic measures such as intravenous immunoglobulins. A significant portion of mature B cell malignancies express B cell receptors (BCR) that use the same immunoglobulin heavy variable gene: IGHV4-34. In particular, ~30% of activated B cell (ABC) diffuse large B cell lymphomas (DLBCL), ~35% of primary central nervous system lymphomas, ~65% of vitreoretinal lymphomas, and ~35% of hairy cell leukemia variant express IGHV4-34. This suggests that the IGHV4-34 heavy chain is critical for driving the disease by delivering cell survival and proliferation signals, and multiple studies have shown that BCR signaling is required for ABC-DLBCL survival. However, while highly enriched in several types of B cell lymphomas, IGHV4-34 expressing B cells compose only ~5% of the normal B cell repertoire of healthy individuals. Therefore, we hypothesized that anti-IGHV4-34 CAR T cells would be highly effective and safe against B cell malignancies, as they would: (i) efficiently recognize IGHV4-34+ lymphoma cells while sparing normal B cells; and (ii) target a tumor driver that is essential for lymphoma cell survival ( Fig 1A). Methods and Results:We developed a novel CAR construct (CD8-41BB-CD3z) targeting the IGHV4-34+ BCR (CART4-34) using a single-chain variable fragment (scFv) derived from the 9G4 rat monoclonal antibody. We used tumor B cells that endogenously (HBL1) or exogenously (Jeko1, Bonna12, Mec1) express the IGHV4-34 BCR. Using cytotoxicity, cytokine secretion and proliferation assays in vitro we showed that CART4-34 specifically target all IGHV4-34+ tumor B cells tested while sparing IGHV4-34- tumor B cells and, most importantly, healthy B cells. However, while the initial CART4-34 showed strong cytotoxicity towards IGHV4-34+ cells in short-term in vitro assays, they were significantly inferior to CART19 in in vivo xenograft models. We hypothesized that the poor performance of CART4-34 compared to CART19 was due to the unique challenge of targeting the membrane-distal portion of the BCR, 18 nm from the plasma membrane. In contrast, CART19 targets an epitope of CD19 located just 5 nm from the membrane ( Fig 1B). Therefore, to improve immune synapse (IS) formation, we designed new CAR constructs with smaller extracellular domains by replacing the original CD8 hinge (44 amino acids (aa)) with either: (i) a short version of the IgG4 hinge (12aa); or (ii) a G4S linker (5aa). We found that both CART4-34 (IgG4) and CART4-34 (G4S) exhibited significantly improved cytotoxicity towards IGHV4-34+ tumor cells in short-term and long-term in vitro assays in all IGHV4-34+ cell lines tested, while maintaining specificity. Importantly, in in vivo human xenograft models of IGHV4-34+ lymphoma (HBL1), CART4-34 with short hinges showed significantly improved tumor control comparable to, or better than, CART19 ( Fig 1B). We also found that while CART19-resistant lymphoma cells had complete loss of CD19 surface expression, CART4-34-resistant tumor cells retained high expression of IgM, suggesting a reduced likelihood of antigen-negative escape owing to the critical role of the BCR for lymphoma B cell survival. Mechanistically, we found that while the original CART4-34 (CD8 hinge) formed IS with striking morphological differences compared to IS formed by CART19, the IS formed by CART4-34 with short hinges highly resembled those formed by CART19, providing a rationale for the improved anti-tumor potency of short-hinge CART4-34. Conclusion:We implemented a novel paradigm for the treatment of B cell malignancies: the specific targeting of the malignant clone, while sparing the remainder of the healthy B cell repertoire. We designed and optimized a best-in-class anti-IGHV4-34 CAR T cell product with potent anti-tumor effects and minimal toxicity towards IGHV4-34-negative B cells. The activity of CART4-34 will be investigated in a Phase I clinical trial.

CXCR4 Loss Reduces the Local Growth of Richter Syndrome Murine and Patient-Derived Xenograft Models and Impairs BCR and VLA-4 Activation

Richter Syndrome (RS) represents transformation of chronic lymphocytic leukemia (CLL) into an aggressive lymphoma and is considered the most important unmet clinical need in CLL because of the lack of effective treatments. An important feature of RS cells is the frequent acquisition of genetic lesions in cell cycle regulators, such as CDKN2A/CDKN2B and TP53, resulting in increased proliferative capacity of the transformed cells and reduced dependence on proliferative microenvironmental signals. However, interactions with the tumor microenvironment are still required to promote the expansion of the transformed cells, as evidenced by the delayed growth of murine TCL1-derived or patient-derived xenograft (PDX) RS models following BCR disruption or macrophage depletion (Chakraborty S et al. Blood 2021, Martines C et al. Blood 2022). To further define the relevance of individual microenvironmental signals in supporting the growth of RS cells in vivo, in this study we evaluated the effects of CRISPR/Cas9-mediated knockout of the chemokine receptor CXCR4, which plays a central role in regulating the homing and interaction with the tumor microenvironment of non-transformed CLL cells. CRISPR/Cas9 editing of CXCR4 was done in the murine TCL1-derived RS model TC1-355 TKO and in the RS-PDX model RS9737, which are both characterized by biallelic disruption of TP53, CDKN2A and CDKN2B. The edited cells, comprising a mixed population with more than 80% disrupted CXCR4 alleles, were then transplanted in the peritoneal cavity and subcutaneous tissue of recipient mice (n=3 C57BL/6 mice for TCL1-355 TKO, n=8 NSG mice for RS9737). Analysis of the mutant allele frequency several weeks after injection revealed a significant reduction in the proportion of cells with mutated CXCR4 in the spleens of the injected mice and to a lesser extent in the peritoneal cavity and subcutaneous tumors, suggesting that disruption of CXCR4 signaling affects not only the migration and homing of the malignant B cells but also their local growth. To further evaluate the latter possibility, we transplanted CXCR4 knockout and CXCR4 wild type RS9737 cells in separate flanks of NSG mice (n=8) and after 4 weeks investigated the percentage of proliferating cells in the tumors by in vivo BrdU incorporation assay. A significantly lower percentage of proliferating cells was observed in the CXCR4 knockout compared to CXCR4 wild type tumors (22.3 ± 2.1 vs 37 ± 1.4, respectively, p&amp;lt;0.001), further suggesting that loss of CXCR4 affects the proliferation of the tumor cells. Since CXCL12 stimulation had no effect on the in vitro proliferation rate of the RS cells, we next investigated whether the reduced in vivo proliferation could be an indirect effect. For this purpose, we investigated whether CXCR4 knockout would affect integrin VLA4 or BCR signaling, as both BCR and CXCR4 can mediate inside-out VLA4 activation and there are data suggesting a potential crosstalk between the BCR and CXCR4 pathways (Maity PC et al. Front Immunol. 2018). Analysis of BCR signaling in the TCL1-355 TKO model showed a significant reduction in anti-IgM induced activation of AKT, ERK and reduced calcium mobilization in the CXCR4 knockout cells despite increased surface IgM expression. However, no effect of CXCR4 knockout on BCR signaling was observed in RS9737 cells, whereas CXCL12-induced VLA4 activation and VCAM1 adhesion were impaired in both models by CXCR4 knockout. To investigate whether impaired VLA4 activation could be responsible for the reduced in vivo proliferation rate of CXCR4 knockout RS cells, we performed BrdU incorporation analysis of RS9737 CXCR4 knockout and CXCR4 wild type cells co-cultured with the human stromal cell line HS5 + CXCL12. Co-culture with HS5 + CXCL12 resulted in a significantly increased proliferation of RS9737 CXCR4 wild type cells compared to the unstimulated control condition (58.5% ± 0.9% vs 50.1% ± 4.3%, respectively, p=0.031), whereas no such difference was observed with RS9737 CXCR4 knockout cells (50.0% ± 1.5% vs 49.1% ± 0.5%, respectively, p=n.s.). Together, these data show that CXCR4 disruption results in negative selection and reduced growth of murine and PDX RS models in vivo and affects BCR and VLA-4 signaling, suggesting that the CXCR4 pathway could represent a therapeutic vulnerability in Richter Syndrome.