IgM Heavy Chain Research Articles

Smoltification of Atlantic Salmon (Salmo salar L.) Is Associated with Enhanced Traffic and Renewal of B Cell Repertoire.

The smoltification of farmed Atlantic salmon is commonly associated with mild immunosuppression. However, B cells may deviate from this trend, showing increased proliferation and migration during this period. This study assessed the effects of smoltification and adaptation to seawater in a controlled experiment. Analyses were conducted on the head kidney, spleen, gill, and both visceral and subcutaneous fat (VAT, SAT) across four time points: parr, early and complete smoltification, and twelve weeks post-seawater transfer. Gene expression analysis was performed to track the distribution and developmental changes in their B cells. Expression profiles of three types of immunoglobulins (ig), including membrane-bound and secreted forms of igm, as well as B cell-specific markers pax1 and cd79, showed strong correlations and contrasted with profiles of other immune cell markers. The highest levels of expression were observed in the lymphatic tissue, followed by the VAT. Enhanced expression in the gill and adipose tissues of smolts suggested an increase in B cell populations. Parallel sequencing of the variable region of the IgM heavy chain was used to track B cell traffic, assessed by the co-occurrence of the most abundant sequences (clonotypes) across different tissues. Smoltification markedly enhanced traffic between all tissues, which returned to initial levels after twelve weeks in the sea. The preferred migration between the head kidney, spleen, and VAT supports the role of abdominal fat as a reservoir of lymphocytes. These findings are discussed in the context of recent studies that suggested the functional significance of B cell traffic in Atlantic salmon. Specifically, the migration of B cells expressing secreted immunoglobulins to virus-infected hearts has been identified as a key factor in the disease recovery and survival of fish challenged with salmon alphavirus (SAV); this process is accelerated by vaccination. Additionally, the study of melanized foci in the skeletal muscles revealed an association between antigen-dependent differentiation and the migration of B cells, indicating a transfer from local to systemic immune responses. Updating the antibody repertoire in the lymphatic and peripheral tissues of smolts may assist in their adaptation to the marine environment and in encountering new pathogens. Emerging evidence highlights B cell migration as an important and previously unrecognized immune mechanism in salmonids.

Effects of a phytogenic diet on growth, haemato-immunological parameters, expression of immune- and stress-related genes, and resistance of Oncorhynchus mykiss to Lactococcus garvieae infection

Phytogenic additives have been implicated in aquafeed owing to their proven growth-stimulating, antimicrobial, antioxidant, and immune-enhancing efficacies. Herein, a short-term feeding experiment (21 days) was designed to explore the effects of a phytogenic additive, PASSIF MOOD® (PM, a mixture of Valeriana officinalis and Passiflora incarnata extracts), on growth, haemato-biochemical variables, immunity, and mRNA expression responses of rainbow trout fingerlings. After feeding, fish were experimentally infected with Lactococcus garvieae and monitored for an additional 20 days. Six groups were fed a commercial diet supplied with different PM levels as 0, 470, 940, 1410, 1880, and 2350 mg/kg and labeled as PM0 (control), PM1, PM2, PM3, PM4, and PM5 groups, respectively. Results revealed significant improvements in the growth of PM-supplied groups compared to the control. Dietary PM significantly increased RBC count, hemoglobin, and hematocrit values compared to those fed on a control diet. Stress indicators such as blood glucose and cortisol levels were significantly decreased in all PM groups than in controls. Moreover, there were significant increases in myeloperoxidase, respiratory burst, and lysozyme activities in PM groups than the control. The highest expression levels of the lysozyme II gene were found in the PM3, PM4, and PM5 groups. Moreover, the mRNA transcripts of il-10 and IgM heavy chain genes were significantly increased in all PM groups compared to the controls. Alternatively, the expression of tnf-α and hsp70 genes were significantly decreased in all PM groups compared to the controls. After challenge, the survival rates in PM0, PM1, PM2, PM3, PM4, and PM5 groups were 37.34%, 84.0%, 85.34%, 68.0%, 64.0%, and 36.0%, respectively, suggesting enhancement of the fish resistance in PM1, PM2, PM3, and PM4 groups compared to control. The results suggest the growth-promoting, antistress, and immunomodulatory roles of the tested phytogenic additive.

Endogenous complement-activating IgM is not required for primary antibody responses but promotes plasma cell differentiation and secondary antibody responses to a large particulate antigen in mice.

Lack of complement factor C1q of the classical pathway results in severely impaired primary antibody responses. This is a paradox because antibodies, especially IgM, are the most efficient activators of the classical pathway and very little specific IgM will be present at priming. A possible explanation would be that natural IgM, binding with low affinity to the antigen, may suffice to activate complement. In support of this, mice lacking secretory IgM have an impaired antibody response, which can be rescued by transfer of non-immune IgM. Moreover, passive administration of specific IgM together with antigen enhances the antibody response in a complement-dependent fashion. To test the idea, we have used a knock-in mouse strain (Cμ13) carrying a point mutation in the IgM heavy chain, rendering the IgM unable to activate complement. Mutant mice backcrossed to BALB/c or C57BL/6 background were primed and boosted with a low dose of sheep red blood cells. Confirming earlier data, no impairment in early, primary IgM- or IgG-responses were seen in either of the Cμ13 strains. However, in one of the mutant strains, late primary IgG responses were impaired. A more pronounced effect was observed after boost, when the IgG response, the number of germinal center B cells and antibody secreting cells as well as the opsonization of antigen were impaired in mutant mice. We conclude that complement activation by natural IgM cannot explain the role of C1q in primary antibody responses, but that endogenous, specific, wildtype IgM generated after immunization feedback-enhances the response to a booster dose of antigen. Importantly, this mechanism can only partially explain the role of complement in the generation of antibody responses because the IgG response was much lower in C3- or complement receptor 1 and 2-deficient mice than in Cμ13 mice.

Peptidylarginine deiminase 2 citrullinates MZB1 and promotes the secretion of IgM and IgA.

MZB1 is an endoplasmic reticulum residential protein preferentially expressed in plasma cells, marginal zone and B1 B cells. Recent studies on murine B cells show that it interacts with the tail piece of IgM and IgA heavy chain and promotes the secretion of these two classes of immunoglobulin. However, its role in primary human B cells has yet to be determined and how its function is regulated is still unknown. The conversion of peptidylarginine to peptidylcitrulline, also known as citrullination, by peptidylarginine deiminases (PADs) can critically influence the function of proteins in immune cells, such as neutrophils and T cells; however, the role of PADs in B cells remains to be elucidated. An unbiased analysis of human lung citrullinome was conducted to identify citrullinated proteins that are enriched in several chronic lung diseases, including rheumatoid arthritis-associated interstitial lung disease (RA-ILD), chronic obstructive pulmonary disease, and idiopathic pulmonary fibrosis, compared to healthy controls. Mass spectrometry, site-specific mutagenesis, and western blotting were used to confirm the citrullination of candidate proteins. Their citrullination was suppressed by pharmacological inhibition or genetic ablation of PAD2 and the impact of their citrullination on the function and differentiation of human B cells was examined with enzyme-linked immunosorbent assay, flow cytometry, and co-immunoprecipitation. Citrullinated MZB1 was preferentially enriched in RA-ILD but not in other chronic lung diseases. MZB1 was a substrate of PAD2 and was citrullinated during the differentiation of human plasmablasts. Ablation or pharmacological inhibition of PAD2 in primary human B cells attenuated the secretion of IgM and IgA but not IgG or the differentiation of IgM or IgA-expressing plasmablasts, recapitulating the effect of ablating MZB1. Furthermore, the physical interaction between endogenous MZB1 and IgM/IgA was attenuated by pharmacological inhibition of PAD2. Our data confirm the function of MZB1 in primary human plasmablasts and suggest that PAD2 promotes IgM/IgA secretion by citrullinating MZB1, thereby contributing to the pathogenesis of rheumatoid arthritis and RA-ILD.

Cold-blooded vertebrates evolved organized germinal center-like structures.

Germinal centers (GCs) or analogous secondary lymphoid microstructures (SLMs) are thought to have evolved in endothermic species. However, living representatives of their ectothermic ancestors can mount potent secondary antibody responses upon infection or immunization, despite the apparent lack of SLMs in these cold-blooded vertebrates. How and where adaptive immune responses are induced in ectothermic species in the absence of GCs or analogous SLMs remain poorly understood. Here, we infected a teleost fish (trout) with the parasite Ichthyophthirius multifiliis (Ich) and identified the formation of large aggregates of highly proliferating IgM+ B cells and CD4+ T cells, contiguous to splenic melanomacrophage centers (MMCs). Most of these MMC-associated lymphoid aggregates (M-LAs) contained numerous antigen (Ag)-specific B cells. Analysis of the IgM heavy chain CDR3 repertoire of microdissected splenic M-LAs and non-M-LA areas revealed that the most frequent B cell clones induced after Ich infection were highly shared only within the M-LAs of infected animals. These M-LAs represented highly polyclonal SLMs in which Ag-specific B cell clonal expansion occurred. M-LA-associated B cells expressed high levels of activation-induced cytidine deaminase and underwent significant apoptosis, and somatic hypermutation of Igμ genes occurred prevalently in these cells. Our findings demonstrate that ectotherms evolved organized SLMs with GC-like roles. Moreover, our results also point to primordially conserved mechanisms by which M-LAs and mammalian polyclonal GCs develop and function.

Sequencing the B cell receptor repertoires of antibody-deficient individuals with and without infection susceptibility

PurposeMost individuals with antibody deficiency (hypogammaglobulinemia) need immunoglobulin replacement therapy (IgG-RT) from healthy plasma donors to stay clear of infections. However, a small subset of hypogammaglobulinemic patients do not require this substitution therapy. We set out to investigate this clinical conundrum by asking whether the peripheral B cell receptor repertoires differ between antibody-deficient patients who do and do not need IgG-RT. MethodsWe sequenced and analyzed IgG and IgM heavy chain B cell receptor repertoires from peripheral blood mononuclear cells (PBMCs) isolated from patients with low serum IgG concentrations who did or did not require IgG-RT. ResultsCompared to the patients who did not need IgG-RT, those who needed IgG-RT had higher numbers of IgG antibody clones, higher IgM diversity, and less oligoclonal IgG and IgM repertoires. The patient cohorts had different heavy chain variable gene usage, and the patients who needed IgG-RT had elevated frequencies of IgG clones with higher germline identity (i.e., fewer somatic hypermutations). ConclusionAntibody-deficient patients with infection susceptibility who needed IgG-RT had more diverse peripheral antibody repertoires that were less diverged from germline and thus may not be as optimal for targeting pathogens, possibly contributing to infection susceptibility.

Moritella viscosa early infection and transcriptional responses of intraperitoneal vaccinated and unvaccinated Atlantic salmon

Skin is the first line of defence against waterborne pathogens, such as Moritella viscosa, which is the causative agent of winter ulcer disease in Atlantic salmon (Salmo salar L.). The present study revealed that vaccination protects against both M. viscosa-induced mortality and skin ulceration, but protection varied according to the strain used in the vaccine formulation. Examination of skin tissue 4 days post M. viscosa challenge indicated that M. viscosa initiated infection by colonising the scale surface. Sequencing the transcripts of the variable region of the IgM heavy chain did not detect effect of vaccination or M. viscosa challenge on antibody repertoire and B cell trafficking from the lymphatic organs to skin. Skin layer-specific responses were examined with 44 K oligonucleotide microarray. Skin responded to vaccination and M. viscosa challenge by increasing transcription of structural genes, stimulating metabolism, and regulation of developmental processes. Immune responses to dead (vaccine) and live M. viscosa were very similar and no pathogen-specific changes were found. Combined use of skin transcriptional profiles and image analysis revealed differences in the infected skin layers between the study groups. M. viscosa was likely retained in the epidermis of vaccinated salmon, whereas the dermis was colonised in unvaccinated fish. Furthermore, decreased expression of lymphocyte-specific and antiviral genes in the dermis indicated possible evasion of immune-related cells from skin in salmon challenged with M. viscosa. Our data also indicated that intraperitoneal vaccination may prime the humoral innate response, which enables a rapid response in the dermis layer of skin during M. viscosa invasion.

Preparation of the monoclonal antibody against Nile tilapia Igλ and study on the Igλ+ B cell subset in Nile tilapia

Immunoglobulins (Igs) are important effector molecules that mediate humoral immunity. A typical Ig consists of two heavy and two light chains. In teleosts, three Ig heavy chain isotypes (Igμ, Igδ and Igτ) and three Ig light chain isotypes (Igκ, Igλ and Igσ) have been identified. Compared to the heavy chains, teleost Ig light chains have been poorly studied due to the lack of antibodies. In this study, a mouse anti-Nile tilapia Igλ monoclonal antibody (mAb) was prepared, which could specifically recognize Igλ in serum and Igλ+ B cells in tissues. Further, the composition of IgM+ and Igλ+ B cell subsets was analyzed using this antibody and a mouse anti-tilapia IgM heavy chain mAb. The ratio of IgM+Igλ+ B cells to total IgM+ B cells in head kidney and peripheral blood was about 30%, while that in spleen was about 50%; the ratio of IgM−Igλ+ B cells to total Igλ+ B cells in head kidney and peripheral blood was about 45%, while that in spleen was about 25%. The IgM−Igλ+ B cells was speculated to be IgT+ B cells. Finally, we detected an increase in the level of specific antibodies against the surface antigen-Sip of Streptococcus agalactiae in serum after S. agalactiae infection, indicating that mouse anti-tilapia Igλ mAb can be used to detect the antibody level after immunization of Nile tilapia, which lays a foundation for the evaluation of immunization effect of tilapia vaccine.

Effects of dietary walnut (Juglans regia) leaves extract on immunity, gene expression responses, and disease resistance in Oreochromis niloticus

The dietary effects of walnut leaf extract (WLE) on the growth, immunity, and resistance of Oreochromis niloticus to bacterial infection have been investigated. Five diets were prepared with various WLE doses of 0, 250, 500, 750, and 1000 mg/kg, termed as Con (control), WLE250, WLE500, WLE750, and WLE1000, respectively. Fish (11.67 ± 0.21 g) were fed these diets for 60 days and then challenged with Plesiomonas shigelloides. Before the challenge, it was observed that dietary WLE did not significantly affect the growth, blood proteins (globulin, albumin, and total protein), and liver function enzymes (ALT and AST) activities. The WLE250 group significantly increased serum SOD and CAT activities more than other groups. The serum immunological indices (lysozyme and myeloperoxidase activities) and hematological parameters (phagocytic activity %, phagocytic index, respiratory burst activity, and potential activity) were significantly increased in the WLE groups compared with the Con group. The expression of IgM heavy chain, IL-1β, and IL-8 genes were significantly upregulated in all WLE-supplemented groups in comparison with the Con group. The fish survival rates (SR; %) post challenge in the Con, WLE250, WLE500, WLE750 and WLE1000 groups were 40.0%, 49.3%, 86.7%, 73.3%, and 70.7%, respectively. The Kaplan–Meier survivorship curves illustrated that the highest SR% was found in the WLE500 group (86.7%) amongst the other groups. Accordingly, we can suggest that feeding O. niloticus with a diet supplied with WLE at a dose rate of 500 mg/kg over 60 days could enrich haemato-immune responses and increase the fish survival against the challenge with P. shigelloides. These results recommend using WLE as a herbal dietary supplement to substitute antibiotic use in aquafeed.

Sequencing the B Cell Receptor Repertoires of Antibody-Deficient Individuals With and Without Infection Susceptibility

PurposeMost individuals with antibody deficiency (hypogammaglobulinemia) need immunoglobulin replacement therapy (IgG-RT) from healthy plasma donors to stay clear of infections. However, a small subset of hypogammaglobulinemic patients do not require this substitution therapy. We set out to investigate this clinical conundrum by asking whether the peripheral B cell receptor repertoires differ between antibody-deficient patients who do and do not need IgG-RT.MethodsWe sequenced and analyzed IgG and IgM heavy chain B cell receptor repertoires from peripheral blood mononuclear cells (PBMCs) isolated from patients with low serum IgG concentrations who did or did not require IgG-RT.ResultsCompared to the patients who did not need IgG-RT, those who needed IgG-RT had higher numbers of IgG antibody clones, higher IgM diversity, and less oligoclonal IgG and IgM repertoires. The patient cohorts had different heavy chain variable gene usage, and the patients who needed IgG-RT had elevated frequencies of IgG clones with higher germline identity (i.e., fewer somatic hypermutations).ConclusionAntibody-deficient patients with infection susceptibility who needed IgG-RT had more diverse peripheral antibody repertoires that were less diverged from germline and thus may not be as optimal for targeting pathogens, possibly contributing to infection susceptibility.

Biochemical, cellular and proteomic profile from head and neck cancer patients submitted to postoperative immunomodulating therapy

Introduction: Head and neck cancer is one the most prevalent forms of cancer in Brazil and worldwide. Immunomodulating enteral nutrition therapy (ENT) can benefit head and neck cancer patients by modulating immune and inflammatory response. Objective: The purpose of this study was to draw a biochemical, cellular and proteomic profile of plasma from head and neck cancer patients submitted to postoperative immunomodulating ENT for 3-5 days. Material and Methods: The cohort was predominantly male, elderly and malnourished. Plasma proteins were quantified and submitted to electrophoresis, depletion chromatography and mass spectrometry. Results: The most highly expressed proteins were C-reactive protein and serum amyloid A1 and A2 (before ENT) and Factor B of the complement system (after ENT). Six protein tumor markers were identified: AAS, plasmin, zinc-α2-glycoprotein, α-1 antichymotrypsin, leucine-rich α-2 glycoprotein 1 and 2, and heavy chain of IgM. Conclusion: In conclusion, the most highly expressed protein in response to postoperative immunomodulating ENT was Factor B of the complement system.

CD123 Directed IgM Antibody-based T-cell Engager, IGM-2537, Demonstrates Potent in vitro and in vivo Activity with Minimal Cytokine Release

Introduction: Despite recent progress, the outlook for patients with acute myeloid leukemia (AML) remains poor. Novel therapies are needed for effective treatment of AML, especially in the relapsed/refractory setting. Evidence suggests that incomplete depletion of leukemia stem cells (LSCs) causes relapse. CD123, the IL-3 receptor alpha chain, is highly expressed on both leukemic blasts and LSCs and thus is a suitable therapeutic target in AML. CD123-based immunotherapies including bispecific antibodies or CAR T cells have shown promise, but cytokine release syndrome (CRS) is a major safety concern with dose limiting toxicity (DLT). We are developing IGM-2537, a novel IgM antibody-based CD123xCD3 bispecific T cell engager (TCE), designed with potent anti-tumor activity and lower cytokine release. IGM-2537 has 10 binding sites for CD123, and single binding site for CD3 via single chain Fv domain (scFv) fused to a joining (J) chain, thus providing high affinity, high avidity binding to CD123-positive malignant cells while co-engagement of CD3 T cells leading to T cell induced cytotoxicity with reduced cytokine release. Here we report functional characterization of IGM-2537 in a broad range of in vitro, ex vivo and in vivo anti-tumor efficacy studies. The improved tolerability, good pharmacodynamic (PD) response and lower cytokine release profile of a benchmark IgM TCE comparator in cynomolgus monkeys will be discussed. Materials and Methods: IGM-2537 was constructed using the pentameric human IgM framework. Recombinant IgM heavy chains and light chains with high affinity variable domains derived from an anti-CD123 IgG were co-expressed with a J-chain fused to anti-CD3 scFv. Surface plasma resonance (SPR), cell membrane protein array, and alanine scanning were utilized to characterize human CD123 binding affinity, specificity, and epitope mapping, respectively. Bispecific enzyme-linked immunosorbent assay (ELISA), T-cell NFAT reporter assay and T-cell dependent cellular cytotoxicity (TDCC) assays were employed to evaluate the potency of IGM-2537 to co-engage both CD123 and CD3 antigens, to induce T cell activation and target cytotoxicity, respectively. Ex vivo AML colony formation assay was applied to assess its activity to eliminate AML blasts. Human MV4-11 xenograft tumor model in humanized NSGdKO mice were utilized to determine anti-tumor efficacy of IGM-2537. Lastly, to confirm that CD123 directed IgM-based TCE molecule can potentially provide better safety profile, a benchmark IgM TCE comparator was assessed in cynomolgus monkeys for tolerability and PK/PD responses. Results and Conclusions: IGM-2537 bound to human CD123 with high affinity, avidity, and specificity. Furthermore, IGM-2537 bound a unique epitope of human CD123 in domain 2 which does not overlap with IL-3. IGM-2537 co-engaged with both CD123 and CD3 antigens, leading to T cell-redirected killing of AML cell lines with concomitant T cell activation. Moreover, IGM-2537 eliminated AML blast cells at physiologically relevant effector/target (E/T) ratios in an ex vivo assay. Most importantly, IGM-2537 demonstrated comparable maximal killing activity to a benchmark IgG TCE molecule but shows significant reduction of cytokine release exemplified by IFN-γ, TNF-α and IL-6, which provide initial evidence of better preclinical safety profile for a CD123 directed IgM-based T-cell engager. As expected, the efficient in vitro TDCC activity of IGM-2537 translated into in vivo anti-tumor efficacy with tumor growth inhibition observed at dose ranges from 1 to 10 mg/kg. To further support that the IgM TCE format can provide a better safety window, a benchmark IgM TCE comparator was evaluated for tolerability (including cytokine production), PK and PD responses in cynomolgus monkeys. All animals tolerated the benchmark comparator well at dose levels up to 10 mg/kg (the maximal dose level evaluated); however, the tolerability observed with IgM TCE was 100x greater compared to IgG TCE. Complete depletion of CD123-positive basophils and substantial reduction of plasmacytoid dendritic cells (pDC) were seen in blood and bone marrow with minimal to no cytokine induction. In summary, our IGM-2537 demonstrates potent in vitro and in vivo activity with minimal cytokine induction. IgM as the framework of a TCE molecule can potentially provide better safety profile and an improved therapeutic window.

Immune profiling of adeno-associated virus response identifies B cell-specific targets that enable vector re-administration in mice.

Adeno-associated virus (AAV) vector-based gene therapies can be applied to a wide range of diseases. AAV expression can last for months to years, but vector re-administration may be necessary to achieve life-long treatment. Unfortunately, immune responses against these vectors are potentiated after the first administration, preventing the clinical use of repeated administration of AAVs. Reducing the immune response against AAVs while minimizing broad immunosuppression would improve gene delivery efficiency and long-term safety. In this study, we quantified the contributions of multiple immune system components of the anti-AAV response in mice. We identified B-cell-mediated immunity as a critical component preventing vector re-administration. Additionally, we found that IgG depletion alone was insufficient to enable re-administration, suggesting IgM antibodies play an important role in the immune response against AAV. Further, we found that AAV-mediated transduction is improved in µMT mice that lack functional IgM heavy chains and cannot form mature B-cells relative to wild-type mice. Combined, our results suggest that B-cells, including non-class switched B-cells, are a potential target for therapeutics enabling AAV re-administration. Our results also suggest that the µMT mice are a potentially useful experimental model for gene delivery studies since they allow repeated dosing for more efficient gene delivery from AAVs.

Dual-Targeting Polymer Nanoparticles Efficiently Deliver DNA Vaccine and Induce Robust Prophylactic Immunity against Spring Viremia of Carp Virus Infection.

ABSTRACTSpring viremia of carp virus (SVCV) is highly contagious and lethal to most cyprinid fish, causing serious economic losses to the carp aquaculture industry. Although DNA vaccines can generate long-term humoral and cellular immune responses, which provide protective immunity against SVCV, the major drawback of DNA vaccines is their low immunogenicity in clinical tests. Here, we construct a dual-targeted polymer DNA vaccine delivery platform (MCS-PCHG) by using mannosylated chitosan to encapsulate the poly(d,l-lactide-co-glycolide)-loaded DNA vaccine containing the heavy-chain CH3 region (CH3) of common carp IgM and the antigenic domain (G131c). The developed nanovaccine delivery platform showed good biocompatibility in vivo and in vitro. With the modification of the mannose moiety and the modification of CH3, the constructed MCS-PCHG could efficiently activate the maturation of antigen-presenting cells. Moreover, we observe significantly high level of immune-related genes expression, serum antigen-specific IgM, SVCV-neutralizing antibody titers in fish vaccinated with MCS-PCHG. Next, the protective efficacy of MCS-PCHG was further evaluated by challenge test. The highest survival rate (ca. 84%) was observed in fish vaccinated with MCS-PCHG after challenging with SVCV. This study presents a novel design for smart, dual-targeted polymer nanoparticles, which are inherently biocompatible, promising for targeted vaccine delivery.IMPORTANCE Spring viremia of carp virus (SVCV) affects global cyprinid fish farming industry, with no available commercial vaccine. Herein, we developed a dual-targeting polymer nanovaccine (MCS-PCHG) by using mannose and common carp IgM heavy chain CH3 region (CH3) as antigen presenting cell (APCs) recognition moiety, attaining the effective delivery of antigen. This dual-targeting polymer vaccine can efficiently activate the APCs, and further induce robust and durable adaptive immune response with good protection against SVCV infection. Our study provides valuable theoretical basis for developing efficient vaccine against infectious diseases in aquaculture.

S135: CRISPR/CAS9 EDITING REVEALS DEPENDENCE OF HUMAN RICHTER SYNDROME AND MURINE CLL CELLS ON SIGNALS FROM B CELL RECEPTOR, CXCR4 RECEPTOR AND MACROPHAGES BUT NOT FROM TOLL-LIKE RECEPTORS IN VIVO

Background: Numerous signals from the microenvironment have been identified that can increase the survival or induce the proliferation of chronic lymphocytic leukemia (CLL) cells in vitro. These signals typically represent various secreted or cell surface ligands that are expressed by different cell types present in the lymph node tumor microenvironment, such as T cells, macrophages and stromal cells, or molecules that would be expected to be released by apoptotic cells, such as apoptosis associated autoantigens or CpG-unmethylated mitochondrial DNA. However, the extent to which these different signals contribute to the growth and survival of the leukemic cells in vivo has still not been fully established. Aims: To determine the relevance of signals from Toll-like receptors (TLRs), the B cell receptor (BCR) and the chemokine receptor CXCR4 for the growth of murine Eμ-TCL1 CLL cells and human Richter Syndrome (RS) patient-derived xenograft (PDX) cells in vivo. Methods: To understand the impact of pharmacological inhibition of the TLR pathway, immunocompetent C57BL/6 or immunodeficient NSG mice were transplanted with murine Eμ-TCL1 CLL or human RS-PDX cells, respectively, and were treated with an inhibitor of the kinase IRAK4 or vehicle control. To determine the effects of genetic disruption of the TLR, BCR and CXCR4 pathways, the IRAK4, IgM heavy chain (IGHM) and CXCR4 genes were disrupted by CRISPR/Cas9 editing in the murine Eμ-TCL1 CLL cells or the human RS-PDX lines RS9737, RS1316 and IP867/17 prior to transplantation. The effects of genetic disruption of these pathways on the growth of the malignant cells in vivo were investigated by analyzing the proportion of mutant and wild type alleles in different anatomical compartments of the transplanted mice. Results: Treatment with the IRAK4 inhibitor significantly prolonged the survival of C57BL/6 mice transplanted with murine Eμ-TCL1 CLL cells and significantly delayed the growth of the human RS-PDX lines RS9737 and RS1316 xenografted in immunodeficient NSG mice. However, genetic disruption of IRAK4 in the human RS-PDX lines RS9737, RS1316 and IP867/17 or of the TLR adaptor MyD88 in the murine Eμ-TCL1 CLL cells did not result in negative selection of these cells in vivo, suggesting that these tumors do not receive or do not rely on TLR signals for their growth and that the therapeutic activity of the IRAK4 inhibitor is not caused by disruption of TLR signaling in the malignant cells themselves. In contrast, genetic disruption of the IGHM or CXCR4 gene was associated with significantly reduced growth of these cells compared to their wild type counterparts and resulted in almost complete disappearance of the mutated cells at later timepoints following transplantation. Analysis of the effects of the IRAK4 inhibitor on other cell types from the tumor microenvironment revealed a significant reduction in the number of macrophages, which in co-culture experiments were shown to strongly protect human Richter syndrome and murine Eμ-TCL1 leukemia cells from spontaneous apoptosis. Summary/Conclusion: These data provide evidence that signals from the BCR, CXCR4 and macrophages support the growth and survival of CLL and RS cells in vivo and argue against a role for TLR signals in the pathogenesis of CLL. In addition, they suggest that targeting the TLR pathway may potentially provide a therapeutic benefit in CLL, but that this benefit would be derived from inhibition of TLR signaling in monocytes and macrophages rather than inhibition of TLR signaling in the malignant cells themselves.

Mystifying Molecular Structure, Expression and Repertoire Diversity of IgM Heavy Chain Genes (Ighμ) in Clarias Catfish and Hybrids: Two Novel Transcripts in Vertebrates.

Two novel immunoglobulin heavy chain (Ighμ) transcripts encoding membrane-bound forms of IgM (mIgM) were discovered in bighead catfish, Clarias macrocephalus. The first transcript contains four constant and two transmembrane domains [Cμ1-Cμ2-Cμ3-Cμ4-TM1-TM2] that have never been reported in teleosts, and the second transcript is an unusual mIgM that has never been identified in any vertebrate [Cμ1-(Cδ2-Cδ3-Cδ4-Cδ5)-Cμ2-Cμ3-TM1-TM2]. Fluorescence in situ hybridization (FISH) in bighead catfish, North African catfish (C. gariepinus) and hybrid catfish revealed a single copy of Ighμ in individual parent catfish, while two gene copies were found in diploid hybrid catfish. Intensive sequence analysis demonstrated multiple distinct structural variabilities in the VH domain in Clarias, and hybrid catfish were defined and used to generate diversity with various mechanisms. Expression analysis of Ighμ in Aeromonas hydrophila infection of the head kidney, peripheral blood leukocytes and spleen revealed significantly higher levels in North African catfish and hybrid catfish than in bighead catfish.

Abstract 3438: Enhanced NK and CD8+ T cell proliferation, tumor cytotoxicity and reversal of T cell exhaustion with IGM-7354, an anti-PD-L1 IgM antibody and IL-15 cytokine fusion

Abstract Anti-PD-1/PD-L1 therapies are efficacious in certain cancer indications, but often patients relapse following a primary response Therefore rational combinations are needed to enhance initial and durable responses of anti-PD-1/PD-L1 therapies. Immunostimulatory cytokine, IL-15 is an attractive combination partner to enhance anti-tumor NK and memory CD8+ T cell expansion and survival. We are developing IGM-7354, a high affinity, high avidity anti-PD-L1 pentameric IgM antibody with an IL-15Rα chain and IL-15 fused to the joining (J) chain, designed to deliver IL-15 to PD-L1 expressing tumors for enhancing anti-tumor immune responses. IGM-7354 was generated by grafting heavy chain variable regions of a high affinity humanized anti-PD-L1 IgG onto the IgM heavy chain framework, co-expressed with the light chain and the J chain which included a single IL-15Rα and IL-15 fusion. Binding ELISAs were performed using recombinant antigens. IGM-7354 bound human and cynomolgus monkey PD-L1 with similar affinities but did not bind to rat or mouse PD-L1. In addition, the IL-15 component of IGM-7354 bound to human and cynomolgus β chain of the trimeric IL-15 receptor with similar affinities, but with weaker binding affinity to rodent IL-15Rβ. Using in vitro assays with human and cynomolgus monkey PBMCs, IGM-7354 dose-dependently enhanced the proliferation of NK and CD8+ T cells. Furthermore, in an in vitro MLR setting, IGM-7354 was able to reverse T cell exhaustion beyond that of an IL-15/IL15Rα complex or anti-PD-L1 IgM or IgG alone, as demonstrated by an increase in activation and effector cytokine secretion. In vitro cytotoxicity and ADCC assays were performed with luciferase-tagged human cancer cell lines and PBMCs. IGM-7354 enhanced in vitro killing of PD-L1-expressing cancer cells by human PBMCs in monotherapy and in combination with other therapeutic agents. Efficacy and pharmacodynamic studies in an MDA-MB-231 xenograft mouse model showed dose-dependent increases in circulating NK and CD8+ T cells and tumor-infiltrating lymphocytes, which correlated with tumor regression. In cynomolgus monkeys, IGM-7354 markedly induced the proliferation of NK and CD8+ T cells in a dose-dependent manner with a preferential expansion of effector memory CD8+ T cells and γδ T cells in the periphery. IGM-7354 stimulates NK and CD8+ T cell expansion in vitro and in vivo plus induces tumor regressions in mouse tumor models in monotherapy or combination with various agents. This approach may enhance tumor localization of the immunostimulatory cytokine IL-15 through high affinity and high avidity binding to PD-L1 thereby improving anti-tumor responses and minimizing toxicity. Citation Format: Thierry Giffon, Melanie Desbois, Poonam Yakkundi, Keerthana Sekar, Marigold Manlusoc, Rodnie Rosete, Daniel Machado, Susan Calhoun, Tasnim Kothambawala, Dean Ng, Vu Tran, Avneesh Saini, Abhinav Jain, Beatrice Wang, Maya Kotturi, Bruce Keyt, Angus Sinclair. Enhanced NK and CD8+ T cell proliferation, tumor cytotoxicity and reversal of T cell exhaustion with IGM-7354, an anti-PD-L1 IgM antibody and IL-15 cytokine fusion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3438.

Assessment of M-protein quantification using capillary electrophoresis and immunosubtraction-based integration in clinical samples with low M-protein concentrations

ObjectivesQuantification of monoclonal protein (M-protein) by serum protein electrophoresis (SPE) is indispensable for diagnosing and monitoring monoclonal gammopathies. However, quantification of small and beta migrating M-proteins is challenging because of overlapping non-immunoglobulin and/or polyclonal immunoglobulin protein fractions. We compared a new integration method based on immunosubtraction (IS-CE) using capillary zone electrophoresis (CZE) against the routine method, which includes a combination of perpendicular drop (0.4%), corrected perpendicular drop (1%) and tangent skimming (98.5%). Design & methodsThe proposed method of M-protein quantification involves calculating the difference in area under the curve between the SPE and a class-specific IS-CE trace. We analyzed the difference in estimated M-protein concentrations obtained with the new method and routine integration methods using 913 samples. For IgA M-proteins at < 10 g/L, the estimated M-protein concentrations were compared with the total IgA concentration. ResultsThe median M-protein concentration of 913 consecutive samples was 6.2 g/L (IQR 2.1–14.3 g/L). The median and median % difference between the two integration methods was 0.68 g/L (IQR 0.01–1.55 g/L) and 10.9% (IQR 0.18–38.7%), showing a larger estimated M-protein concentration with the new method. More than 25% difference was observed in 38% of the samples and was associated with lower total protein concentration, lower M-protein concentration, IgA and IgM heavy chain isotypes, and beta- or beta-gamma migration. When 161 samples with IgA M-protein < 10 g/L were compared against total IgA concentration, the median bias of the new method was smaller compared to that of the existing method (-0.95 g/L vs. −1.3 g/L, P < 0.0001). ConclusionsThe use of IS based integration using CZE and IS-CE is promising especially for small and beta migrating M-proteins.

Criteria for Presumptive Diagnosis of IgD Multiple Myeloma

IgD myelomas constitute about 1% of myelomas, have worse prognosis, have higher association with lambda light chains, and are associated with excess free involved light chains. Standard immunofixation protocols stain only for IgG, IgM and IgA heavy chains. Proper attribution of a monoclonal gammopathy as having a delta heavy chain requires additional testing that is not available in most clinical laboratories. Retrospective review of protein electrophoresis and other laboratory results showed characteristic findings in IgD myelomas.

Characterization of human IgM and IgG repertoires in individuals with chronic HIV-1 infection.

Advancements in high-throughput sequencing (HTS) of antibody repertoires (Ig-Seq) have unprecedentedly improved our ability to characterize the antibody repertoires on a large scale. However, currently, only a few studies explored the influence of chronic HIV-1 infection on human antibody repertoires and many of them reached contradictory conclusions, possibly limited by inadequate sequencing depth and throughput. To better understand how HIV-1 infection would impact humoral immune system, in this study, we systematically analyzed the differences between the IgM (HIV-IgM) and IgG (HIV-IgG) heavy chain repertoires of HIV-1 infected patients, as well as between antibody repertoires of HIV-1 patients and healthy donors (HH). Notably, the public unique clones accounted for only a negligible proportion between the HIV-IgM and HIV-IgG repertoires libraries, and the diversity of unique clones in HIV-IgG remarkably reduced. In aspect of somatic mutation rates of CDR1 and CDR2, the HIV-IgG repertoire was higher than HIV-IgM. Besides, the average length of CDR3 region in HIV-IgM was significant longer than that in the HH repertoire, presumably caused by the great number of novel VDJ rearrangement patterns, especially a massive use of IGHJ6. Moreover, some of the B cell clonotypes had numerous clones, and somatic variants were detected within the clonotype lineage in HIV-IgG, indicating HIV-1 neutralizing activities. The in-depth characterization of HIV-IgG and HIV-IgM repertoires enriches our knowledge in the profound effect of HIV-1 infection on human antibody repertoires and may have practical value for the discovery of therapeutic antibodies.