Genetic Characteristics of Non B Cell-Derived Immunoglobulin Genes.

It is widely acknowledged that immunoglobulins (Igs) are produced solely by B-lineage cells. The Ig gene is created by the rearrangement of a group of gene segments [variable (V), diversity (D), and joining (J) segments rearrangement, or V(D)J recombination], which results in the vast diversity of B cell-derived Ig responsible for recognising various antigens. Ig subsequently undergoes somatic hypermutation (SHM) and class switch recombination (CSR) after exposure to antigens, thus converting the low-affinity IgM to IgG, IgA, or IgE antibodies. IgM and IgD are primarily expressed in naïve B cells that have not been exposed to antigens, they do not undergo somatic hypermutation; hence, their variable region sequences remain the same as those in the germline. In contrast, IgG, IgA, and IgE are expressed in antigen-stimulated memory B cells or plasma cells, and thus, they often possess high-frequency mutations in their variable region sequences. Since the discovery that Ig can be produced by non-B cells, Qiu's group has investigated and compared the genetic characteristics of B cell-derived Ig and non-B cell-derived Ig. These findings demonstrated that non-B cell-derived Ig shares certain similarities with B cell-derived Ig in that the sequence of its constant region is identical to that of B cell-derived Ig, and its variable region is also strictly dependent on the rearrangement of V, D, and J gene segments. Moreover, akin to B cell-derived Ig, the V regions of IgM and IgD are rarely mutated, while IgG, IgA, and IgE produced by cancer cells are frequently mutated. However, the non-B cell-derived Ig V region sequence displays unique characteristics. (1) Unlike the vast diversity of B cell-derived Igs, non-B cell-derived Igs exhibit restricted diversity; cells from the same lineage always select the same V(D)J recombination patterns; (2) Both mRNA and proteins of RAG1/RAG2 recombinase have been detected in Ig positive cancer cell lines and normal tissues. But Ig recombination could also be found in RAG1-/- and RAG2-/- mice, suggesting that they are not necessary for the rearrangement of non-B cell-derived Igs. These features of non-B cell-derived Igs suggest a potentially undiscovered mechanism of V(D)J recombination, ligation, and SHM in non-B cells, which necessitates further investigation with advanced technology in molecular biology.

Expression, Function, and Significance of Non B Cell-Derived Immunoglobulin in Haematological System.

Acute myeloid leukaemia (AML) is a collection of genetically diverse diseases characterised by abnormal proliferation of immature haematopoietic cells and disruption of normal haematopoiesis. Myeloid cells and lymphocytes originate from different haematopoietic precursors within the bone marrow. It has been traditionally assumed that myeloid cells cannot produce immunoglobulin (Ig), a marker of B cells and plasma cells. However, in recent years, all five Ig classes have been detected in CD34+ haematopoietic stem cells, mature monocytes and neutrophils, differentiated macrophages and tumour-associated macrophages, acute myeloid leukaemia cell lines, as well as myeloblasts of AML. The rearranged V(D)J sequences exhibit unique restricted or biased V gene usage and evidence of somatic mutation. Furthermore, AML-derived Igs could promote cell proliferation, induce apoptosis, and enhance migration. Elevated levels of Ig expression predict inferior clinical outcomes. These findings indicate that AML-derived Ig plays a role in AML pathogenesis and progression, and could serve as a novel biomarker for risk stratification, disease monitoring, and targeted therapy. In this chapter, we provide a comprehensive review of recent literature on the expression, function, and significance of non B cell-derived Ig in the haematological system, with a focus on AML.

High levels of immunoglobulin expression predict shorter overall survival in patients with acute myeloid leukemia.

OBJECTIVES: It has been believed that immunoglobulins can only be produced by B lymphocytes and plasma cells. We have previously reported that IgG can be expressed in myeloblasts from patients with acute myeloid leukemia (AML) and plays a role in the proliferation and apoptosis of leukemic cells. However, its clinical impact has not been assessed. METHODS: We assessed the expression of different classes of immunoglobulin in peripheral blood and bone marrow samples from 132 AML patients and correlated the levels of expression with clinicopathologic and molecular genetic features, as well as clinical outcome. RESULTS: We found that, in addition to IgG, all classes of immunoglobulin are expressed in myeloblasts, including IgG, IgM, IgA, IgD, IgE, Igκ, and Igλ. The levels of IgG expression (coupled with Igκ or Igλ) are higher than those of IgM, IgA, IgD, and IgE. Using receiver operating characteristic (ROC) curve analysis, we identified two distinct groups of AML patients with differential expression of immunoglobulin and different clinical outcomes. CONCLUSIONS: High levels of immunoglobulin expression are associated with monocytic differentiation, multilineage dysplasia, TET2 and KRAS mutations, and poor overall survival. Assessment of immunoglobulin may serve as a useful marker for prognostic stratification and target therapy.

Lsd1 safeguards T-cell development via suppressing endogenous retroelements and interferon responses.

The histone demethylase Lsd1 has been shown to play multiple essential roles in mammalian biology. However, its physiological functions in thymocyte development remain elusive. We observed that the specific deletion of Lsd1 in thymocytes caused significant thymic atrophy and reduced peripheral T cell populations with impaired proliferation capacity. Single-cell RNA sequencing combined with strand-specific total RNA-seq and ChIP-seq analysis revealed that ablation of Lsd1 led to the aberrant derepression of endogenous retroelements, which resulted in a viral mimicry state and activated the interferon pathway. Furthermore, the deletion of Lsd1 blocked the programmed sequential down-regulation of CD8 expression at the DP→CD4+CD8lo stage and induced an innate memory phenotype in both thymic and peripheral T cells. Single-cell TCR sequencing revealed the kinetics of TCR recombination in the mouse thymus. However, the preactivation state after Lsd1 deletion neither disturbed the timeline of TCR rearrangement nor reshaped the TCR repertoire of SP cells. Overall, our study provides new insight into the function of Lsd1 as an important maintainer of endogenous retroelement homeostasis in early T-cell development.

Next-Generation Sequencing Revealed a Distinct Immunoglobulin Repertoire with Specific Mutation Hotspots in Acute Myeloid Leukemia.

Immunoglobulin (Ig) is known as a hallmark of B-lymphocytes exerting antibody functions. However, our previous studies demonstrated that myeloblasts from acute myeloid leukemia (AML) patients could also express Ig with distinct roles. Here, we quantified Ig (IGHG and IGK) transcripts by real-time PCR and performed a comprehensive analysis of Ig repertoire (both heavy chains and light chains) in AML blasts. We found that Ig was frequently expressed by AML blasts. A higher level of AML-derived IGHG expression correlated with a significantly shorter disease-free survival. Next-generation sequencing revealed dysregulated transcripts of all five Ig classes (IGHA, IGHD, IGHE, IGHG, and IGHM) and two Ig types (IGK and IGL) in AML. VH-D-JH rearrangements in myeloblasts were biased with individual specificity rather than generally diverse as in B-cells. Compared to AML-derived IgH, AML-derived IGK was more conserved among different AML samples. The frequently shared Vκ-Jκ patterns were IGKV3-20*01/IGKJ1*01, IGKV2D-28*01/IGKJ1*01, and IGKV4-1*01/IGKJ1*01. Moreover, AML-derived IGK was different from classical IGK in B-cells for the high mutation rates and special mutation hotspots at serine codons. Findings of the distinct Ig repertoire in myeloblasts may facilitate the discovery of a new molecular marker for disease monitoring and target therapy.

Epigenetic Regulation of NK Cell-Mediated Antitumor Immunity.

Natural killer (NK) cells are critical innate lymphocytes that can directly kill target cells without prior immunization. NK cell activation is controlled by the balance of multiple germline-encoded activating and inhibitory receptors. NK cells are a heterogeneous and plastic population displaying a broad spectrum of functional states (resting, activating, memory, repressed, and exhausted). In this review, we present an overview of the epigenetic regulation of NK cell-mediated antitumor immunity, including DNA methylation, histone modification, transcription factor changes, and microRNA expression. NK cell-based immunotherapy has been recognized as a promising strategy to treat cancer. Since epigenetic alterations are reversible and druggable, these studies will help identify new ways to enhance NK cell-mediated antitumor cytotoxicity by targeting intrinsic epigenetic regulators alone or in combination with other strategies.

Histone methyltransferase Ezh2 negatively regulates NK cell terminal maturation and function.

NK cells are innate lymphoid cells that play important roles in tumor eradication and viral clearance. We previously found that deletion or inhibition of the histone methyltransferase Ezh2 (enhancer of zeste homolog 2) in hematopoietic stem and progenitor cells (HSPCs) from both mice and humans enhanced the commitment and cytotoxicity of NK cells to tumor cells. This study tested the hypothesis that inhibiting Ezh2, especially in NK lineage cells, could also affect NK cell development and function. We crossed Ezh2fl/fl mice with Ncr1iCre mice to delete the Ezh2 gene in immature NK cells and downstream progeny. Ezh2 deficiency increased the total number of NK cells and promoted NK cell terminal differentiation, as the percentages of the most mature CD27- CD11b+ subsets were increased. The NK cell cytotoxicity against tumor cells in vitro was enhanced, with increased degranulation and IFN-γ production. In addition, during the process of human NK cells differentiating from HSPCs , inhibiting EZH2 catalytic activity at day 14 (when NK lineage commitment began) also resulted in increased proportions of mature NK cells and cytotoxicity. Furthermore, RNA-seq and CUT&RUN-qPCR assays showed that the effects of Ezh2 may be based on its direct modulation of the expression of the transcription factor Pbx1 (pre-B-cell leukemia transcription factor 1), which has been reported to promote NK cell development. In summary, we demonstrate that Ezh2 is a negative regulator of NK cell terminal maturation and function.

Functional analysis of BRCT missense mutations in BRCA1-mutated Chinese Han familial breast cancer.

Breast cancer 1 (BRCA1) is one of the most common tumor suppressor genes in breast cancer. The BRCT domain of BRCA1 has been shown to have a critical role in tumor suppression. In a previous study, two de novo BRCT missense mutations of BRCA1, G1763V and L1786P were identified from Chinese females with familial breast cancer. In the present study, the function of these two novel mutations were assessed by bioinformatics analysis and a series of experiments investigating cell proliferation, cell cycle and chemotherapy combination. Although bioinformatics analysis indicated that the mutants may be deleterious, a series of experiments revealed that the two mutants significantly reduced the growth and increased cell apoptosis similar to the function of BRCA1 wild type. Furthermore, no synergistic effect between the Olaparib and BRCA1 mutation was noted on cell apoptosis. These results demonstrated that these two mutations did not affect the tumor suppressor function of BRCA1. It was concluded that not all BRCA1 missense mutations are pathogenic and that any new BRCA1 mutation should be assessed for its effect on the tumor suppressor function of BRCA1.

Immunoglobulin gene expression in umbilical cord blood-derived CD34⁺ hematopoietic stem/progenitor cells.

Recently, immunoglobulin (Ig) expression was reported in a variety of non-B lineage cells, including myeloid cells. We assessed whether hematopoietic stem/progenitor cells (HSC/HPCs) can express Ig. With Gene Expression Omnibus (GEO) microarray database analysis, we found that IGHM was expressed with the highest frequency and level in umbilical cord blood CD34(+) HSC/HPCs, followed by IGK@, IGHE, IGHD, IGHG1, and IGHA1, while IGL@ was nearly not expressed. Ig expression was further confirmed by molecular experiments and immunofluorescence. Moreover, HSC/HPCs-derived Ig displayed restricted/biased usages and VHDJH rearrangement patterns. These results suggest that Igs, especially IgM, may have a role in CD34(+) HSC/HPCs function.

IGK with conserved IGΚV/IGΚJ repertoire is expressed in acute myeloid leukemia and promotes leukemic cell migration.

We have previously reported that immunoglobulin heavy chain genes were expressed in myeloblasts and mature myeloid cells. In this study, we further demonstrated that rearranged Ig κ light chain was also frequently expressed in acute myeloid leukemia cell lines (6/6), primary myeloblasts from patients with acute myeloid leukemia (17/18), and mature monocytes (11/12) and neutrophils (3/12) from patients with non-hematopoietic neoplasms, but not or only rarely expressed in mature neutrophils (0/8) or monocytes (1/8) from healthy individuals. Interestingly, myeloblasts and mature monocytes/neutrophils shared several restricted IGKV and IGKJ gene usages but with different expression frequency. Surprisingly, almost all of the acute myeloid leukemia-derived IGKV showed somatic hypermutation; in contrast, mature myeloid cells-derived IGKV rarely had somatic hypermutation. More importantly, although IGK expression appeared not to affect cell proliferation, reduced IGK expression led to a decrease in cell migration in acute myeloid leukemia cell lines HL-60 and NB4, whereas increased IGK expression promoted their motility. In summary, IGK is expressed in myeloblasts and mature myeloid cells from patients with non-hematopoietic neoplasms, and is involved in cell migration. These results suggest that myeloid cells-derived IgK may have a role in leukemogenesis and may serve as a novel tumor marker for monitoring minimal residual disease and developing target therapy.