Longitudinal analysis of FcRL5 expression and clonal relationships among classical and atypical memory B cells following malaria.

BACKGROUND: Chronic and frequently recurring infectious diseases, such as malaria, are associated with expanded populations of atypical memory B cells (MBCs). These cells are different from classical MBCs by the lack of surface markers CD21 and CD27 and increased expression of inhibitory receptors, such as FcRL5. While the phenotype and conditions leading to neogenesis of atypical MBCs in malaria-experienced individuals have been studied extensively, the origin of these cells remains equivocal. Functional similarities between FcRL5+ atypical MBCs and FcRL5+ classical MBCs have been reported, suggesting that these cells may be developmentally related. METHODS: Here, a longitudinal analysis of FcRL5 expression in various B cell subsets was performed in two children from a high transmission region in Uganda over a 6-month period in which both children experienced a malaria episode. Using B-cell receptor (BCR)-sequencing to track clonally related cells, the connections between IgM+ and IgG+ atypical MBCs and other B cell subsets were studied. RESULTS: The highest expression of FcRL5 was found among IgG+ atypical MBCs, but FcRL5+ cells were present in all MBC subsets. Following malaria, FcRL5 expression increased in all IgM+ MBC subsets analysed here: classical, activated, and atypical MBCs, while results for IgG+ MBC subsets were inconclusive. IgM+ atypical MBCs showed few connections with other B cell subsets, higher turnover than IgG+ atypical MBCs, and were predominantly derived from naïve B cells and FcRL5- IgM+ classical MBCs. In contrast, IgG+ atypical MBCs were clonally expanded and connected with classical MBCs. IgG+ atypical MBCs present after a malaria episode mainly originated from FcRL5+ IgG+ classical MBCs. CONCLUSIONS: Collectively, these results suggest fundamental differences between unswitched and class-switched B cell populations and provide clues about the primary developmental pathways of atypical MBCs in malaria-experienced individuals.

A Molecular Analysis of Memory B Cell and Antibody Responses Against Plasmodium falciparum Merozoite Surface Protein 1 in Children and Adults From Uganda.

Memory B cells (MBCs) and plasma antibodies against Plasmodium falciparum (Pf) merozoite antigens are important components of the protective immune response against malaria. To gain understanding of how responses against Pf develop in these two arms of the humoral immune system, we evaluated MBC and antibody responses against the most abundant merozoite antigen, full-length Pf merozoite surface protein 1 (PfMSP1FL), in individuals from a region in Uganda with high Pf transmission. Our results showed that PfMSP1FL-specific B cells in adults with immunological protection against malaria were predominantly IgG+ classical MBCs, while children with incomplete protection mainly harbored IgM+ PfMSP1FL-specific classical MBCs. In contrast, anti-PfMSP1FL plasma IgM reactivity was minimal in both children and adults. Instead, both groups showed high plasma IgG reactivity against PfMSP1FL, with broadening of the response against non-3D7 strains in adults. The B cell receptors encoded by PfMSP1FL-specific IgG+ MBCs carried high levels of amino acid substitutions and recognized relatively conserved epitopes on the highly variable PfMSP1 protein. Proteomics analysis of PfMSP119-specific IgG in plasma of an adult revealed a limited repertoire of anti-MSP1 antibodies, most of which were IgG1 or IgG3. Similar to B cell receptors of PfMSP1FL-specific MBCs, anti-PfMSP119 IgGs had high levels of amino acid substitutions and their sequences were predominantly found in classical MBCs, not atypical MBCs. Collectively, these results showed evolution of the PfMSP1-specific humoral immune response with cumulative Pf exposure, with a shift from IgM+ to IgG+ B cell memory, diversification of B cells from germline, and stronger recognition of PfMSP1 variants by the plasma IgG repertoire.

B Cell Receptor Repertoire Analysis in Malaria-Naive and Malaria-Experienced Individuals Reveals Unique Characteristics of Atypical Memory B Cells.

Malaria, caused by parasites of the Plasmodium genus, is responsible for significant morbidity and mortality globally. Chronic Plasmodium falciparum exposure affects the B cell compartment, leading to the accumulation of atypical memory B cells (atMBCs). IgM-positive (IgM+) and IgG+ atMBCs have not been compared in-depth in the context of malaria, nor is it known if atMBCs in malaria-experienced individuals are different from phenotypically similar B cells in individuals with no known history of Plasmodium exposure. To address these questions, we characterized the B cell receptor (BCR) repertoire of naive B cells (NBCs), IgM+ and IgG+ classical MBCs (cMBCs), and IgM+ and IgG+ atMBCs from 13 malaria-naive American adults and 7 malaria-experienced Ugandan adults. Our results demonstrate that P. falciparum exposure mainly drives changes in atMBCs. In comparison to malaria-naive adults, the BCR repertoire of Plasmodium-exposed adults showed increased levels of somatic hypermutation in the heavy chain V region in IgM+ and IgG+ atMBCs, shorter heavy chain complementarity-determining region 3 (HCDR3) in IgG+ atMBCs, and increased usage of IGHV3-73 in IgG+ cMBCs and both IgM+ and IgG+ atMBCs. Irrespective of Plasmodium exposure, IgM+ atMBCs closely resembled NBCs, while IgG+ atMBCs resembled IgG+ cMBCs. Physicochemical properties of the HCDR3 seemed to be intrinsic to cell type and independent of malaria experience. The resemblance between atMBCs from Plasmodium-exposed and naive adults suggests similar differentiation pathways regardless of chronic antigen exposure. Moreover, these data demonstrate that IgM+ and IgG+ atMBCs are distinct populations that should be considered separately in future analyses. IMPORTANCE Malaria, caused by Plasmodium parasites, still contributes to a high global burden of disease, mainly in children under 5 years of age. Chronic and recurrent Plasmodium infections affect the development of B cell memory against the parasite and promote the accumulation of atypical memory B cells (atMBCs), which have an unclear function in the immune response. Understanding where these cells originate from and whether they are beneficial in the immune response to Plasmodium will help inform vaccination development efforts. We found differences in B cell receptor (BCR) properties of atMBCs between malaria-naive and malaria-experienced adults that are suggestive of divergent selection processes, resulting in more somatic hypermutation and differential immunoglobulin heavy chain V (IGHV) gene usage. Despite these differences, atMBCs from malaria-naive and malaria-experienced adults also showed many similarities in BCR characteristics, such as physicochemical properties of the HCDR3 region, suggesting that atMBCs undergo similar differentiation pathways in response to different pathogens. Our study provides new insights into the effects of malaria experience on the B cell compartment and the relationships between atMBCs and other B cell populations.

Potential functions of atypical memory B cells in Plasmodium-exposed individuals.

Naturally acquired iummunity against clinical malaria is slow to develop, taking years of repeated exposure to parasites to acquire sufficiently broad and potent antibody responses. Increasing evidence suggests that Plasmodium infection and the resulting immune stimulation contribute to changes in the B cell compartment. In particular, accumulation of atypical memory B cells (atMBCs) is common in Plasmodium-exposed individuals. Similarities to B cell subsets present in other acute and chronic disease settings have provided insight into the development and potential function of these cells; however, their contribution to protection against malaria is still poorly understood. Here, we discuss recent findings that have increased our understanding of atMBCs and outline outstanding questions related to their function and development in the protective immune response to malaria.

Naturally Acquired Humoral Immunity Against Plasmodium falciparum Malaria.

Malaria remains a significant contributor to the global burden of disease, with around 40% of the world's population at risk of Plasmodium infections. The development of an effective vaccine against the malaria parasite would mark a breakthrough in the fight to eradicate the disease. Over time, natural infection elicits a robust immune response against the blood stage of the parasite, providing protection against malaria. In recent years, we have gained valuable insight into the mechanisms by which IgG acts to prevent pathology and inhibit parasite replication, as well as the potential role of immunoglobulin M (IgM) in these processes. Here, we discuss recent advances in our understanding of the mechanisms, acquisition, and maintenance of naturally acquired immunity, and the relevance of these discoveries for the development of a potential vaccine against the blood stage of Plasmodium falciparum.

NCL Inhibition Exerts Antineoplastic Effects against Prostate Cancer Cells by Modulating Oncogenic MicroRNAs.

Prostate cancer (PCa) is the most frequently diagnosed cancer in men and second most common cause of cancer-related deaths in the United States. Androgen deprivation therapy (ADT) is only temporarily effective for advanced-stage PCa, as the disease inevitably progresses to castration-resistant prostate cancer (CRPC). The protein nucleolin (NCL) is overexpressed in several types of human tumors where it is also mislocalized to the cell surface. We previously reported the identification of a single-chain fragment variable (scFv) immuno-agent that is able to bind NCL on the surface of breast cancer cells and inhibit proliferation both in vitro and in vivo. In the present study, we evaluated whether NCL could be a valid therapeutic target for PCa, utilizing DU145, PC3 (CRPC), and LNCaP (androgen-sensitive) cell lines. First, we interrogated the publicly available databases and noted that higher NCL mRNA levels are associated with higher Gleason Scores as well as with recurrent and metastatic tumors. Then, using our anti-NCL scFv, we demonstrated that NCL is expressed on the surface of all three tested cell lines and that NCL inhibition results in reduced proliferation and migration. We also measured the inhibitory effect of NCL targeting on the biogenesis of oncogenic microRNAs such as miR-21, -221 and -222, which was cell context dependent. Taken together, our data provide evidence that NCL targeting inhibits the key hallmarks of malignancy in PCa cells and may provide a novel therapeutic option for patients with advanced-stage PCa.

A novel fully human anti-NCL immunoRNase for triple-negative breast cancer therapy.

Breast cancer is the most common cancer in women worldwide. A new promising anti-cancer therapy involves the use of monoclonal antibodies specific for target tumor-associated antigens (TAAs). A TAA of interest for immunotherapy of Triple Negative Breast Cancer (TNBC) is nucleolin (NCL), a multifunctional protein, selectively expressed on the surface of cancer cells, which regulates the biogenesis of specific microRNAs (miRNAs) involved in tumor development and drug-resistance. We previously isolated a novel human anti-NCL scFv, called 4LB5, that is endowed with selective anti-tumor effects. Here we report the construction and characterization of a novel immunoRNase constituted by 4LB5 and a human pancreatic RNase (HP-RNase) called "4LB5-HP-RNase". This immunoRNase retains both the enzymatic activity of human pancreatic RNase and the specific binding of the parental scFv to a panel of surface NCL-positive breast cancer cells. Notably, 4LB5-HP-RNase dramatically and selectively reduced the viability and proliferation of NCL-positive tumor cells in vitro and in vivo. Specifically, it induced apoptosis and reduced the levels of the tumorigenic miRNAs miR-21, -221 and -222. Thus, this novel immunoagent could be a valuable tool for the treatment of TNBC patients ineligible for currently available targeted treatments.

Ran Binding Protein 9 (RanBP9) is a novel mediator of cellular DNA damage response in lung cancer cells.

Ran Binding Protein 9 (RanBP9, also known as RanBPM) is an evolutionary conserved scaffold protein present both in the nucleus and the cytoplasm of cells whose biological functions remain elusive. We show that active ATM phosphorylates RanBP9 on at least two different residues (S181 and S603). In response to IR, RanBP9 rapidly accumulates into the nucleus of lung cancer cells, but this nuclear accumulation is prevented by ATM inhibition. RanBP9 stable silencing in three different lung cancer cell lines significantly affects the DNA Damage Response (DDR), resulting in delayed activation of key components of the cellular response to IR such as ATM itself, Chk2, γH2AX, and p53. Accordingly, abrogation of RanBP9 expression reduces homologous recombination-dependent DNA repair efficiency, causing an abnormal activation of IR-induced senescence and apoptosis. In summary, here we report that RanBP9 is a novel mediator of the cellular DDR, whose accumulation into the nucleus upon IR is dependent on ATM kinase activity. RanBP9 absence hampers the molecular mechanisms leading to efficient repair of damaged DNA, resulting in enhanced sensitivity to genotoxic stress. These findings suggest that targeting RanBP9 might enhance lung cancer cell sensitivity to genotoxic anti-neoplastic treatment.