IgA- Tertiary Lymphoid Structures Correlates With a Better Prognosis in GADC Patients

Background: The roles of tumor infiltrating B lymphocytes (TIBs) and tertiary lymphoid structures (TLSs) in solid tumor genesis and tumor therapy have been recognized by researchers, but the specific formation and effect of the TLSs have not been fully understood. In this study, we used single-cell RNA sequencing, multiple immunofluorescence assays, and quantitative digital image analysis to study the formation and structure of TLSs in gastric adenocarcinoma (GADC). Furthermore, the study collected 165 cases of GADC with TLSs and analysis the relationships between TLS formation and clinicopathological characteristics and prognosis of GADC patients were analyzed. Results:The result identified the type of IgA-TLSs which contained higher level of IgA+-B cells in GADC, and the major structures of the IgA-TLSs were determined. We found that immune cells in IgA-TLSs had higher levels of cellular interactions and migration ability. The expression of signal sequence receptor subunit 4 (SSR4) was characterized and found to higher expressed in the IgA-TLSs. Furthermore, IgA-TLSs correlated with age, differentiation, distant metastasis, TNM stage, chemotherapy effect, expression of programmed death-ligand 1, J-chain, and SSR4, and better overall survival. Conclusions: Our research provided the information about CD79A/J-chain B cells in GADC and indicated that IgA-TLSs was associated with better prognosis for GADC patients.

Role of Polymeric Immunoglobulin Receptor in IgA and IgM Transcytosis

Transcytosis of polymeric IgA and IgM from the basolateral surface to the apical side of the epithelium and subsequent secretion into mucosal fluids are mediated by the polymeric immunoglobulin receptor (pIgR). Secreted IgA and IgM have vital roles in mucosal immunity in response to pathogenic infections. Binding and recognition of polymeric IgA and IgM by pIgR require the joining chain (J chain), a small protein essential in the formation and stabilization of polymeric Ig structures. Recent studies have identified marginal zone B and B1 cell-specific protein (MZB1) as a novel regulator of polymeric IgA and IgM formation. MZB1 might facilitate IgA and IgM transcytosis by promoting the binding of J chain to Ig. In this review, we discuss the roles of pIgR in transcytosis of IgA and IgM, the roles of J chain in the formation of polymeric IgA and IgM and recognition by pIgR, and focus particularly on recent progress in understanding the roles of MZB1, a molecular chaperone protein.

Detailed Structure and Pathophysiological Roles of the IgA-Albumin Complex in Multiple Myeloma

Immunoglobulin A (IgA)-albumin complexes may be associated with pathophysiology of multiple myeloma, although the etiology is not clear. Detailed structural analyses of these protein–protein complexes may contribute to our understanding of the pathophysiology of this disease. We analyzed the structure of the IgA-albumin complex using various electrophoresis, mass spectrometry, and in silico techniques. The data based on the electrophoresis and mass spectrometry showed that IgA in the sera of patients was dimeric, linked via the J chain. Only dimeric IgA can bind to albumin molecules leading to IgA-albumin complexes, although both monomeric and dimeric forms of IgA were present in the sera. Molecular interaction analyses in silico implied that dimeric IgA and albumin interacted not only via disulfide bond formation, but also via noncovalent bonds. Disulfide bonds were predicted between Cys34 of albumin and Cys311 of IgA, resulting in an oxidized form of albumin. Furthermore, complex formation prolongs the half-life of IgA molecules in the IgA-albumin complex, leading to excessive glycation of IgA molecules and affects the accumulation of IgA in serum. These findings may demonstrate why complications such as hyperviscosity syndrome occur more often in patients with IgA dimer producing multiple myeloma.

569 Targeting IL-15 delivery to PD-L1 expressing tumors with an Anti-PD-L1-IL-15 cytokine fusion IgM to enhance T Cell and NK Cell Mediated tumor cytotoxicity

BackgroundTherapeutic antibodies inhibiting PD-1/PD-L1 have demonstrated clinical efficacy though only a fraction of patients respond. Combinations are being explored to enhance responses including anti-PD-1/PD-L1 IgG antibodies with IL-15-pathway stimulating agents to remove PD-1 immunosuppressive signaling and enhance anti-tumor NK and memory CD8 T cell expansion and survival. We have engineered an anti-PD-L1 pentameric high affinity, high avidity IgM, to target low PD-L1 expressing tumors, with an IL-15 superagonist fused to the joining (J) chain.MethodsAn anti-PD-L1 IgM was generated by grafting heavy chain variable regions of a high affinity IgG onto the IgM heavy chain framework and co-expressed with the light chains. The IL-15 superagonist fused to the J chain generated PDL1-ISA. Anti-PD-L1 binding was performed using recombinant antigen ELISAs and on cells by FACS. Reporter assays and PBMCs were used for potency testing. Cytokines were evaluated by CBA assays. In vitro cytotoxicity assays used luciferase tagged MDA-MB-231 cells with PBMCs, NK or CD8 T cells. Pharmacodynamic and efficacy studies were conducted in syngeneic and humanized mouse models.ResultsThe parental anti-PD-L1 IgM antibody bound recombinant and cellular PD-L1 more potently than an IgG antibody with the same binding domain. In functional PD-L1 and PD-1 blocking studies the anti-PD-L1 IgM was as efficacious as the IgG. PDL1-ISA provided a potent proliferation signal to primary human NK and CD8 T cells in vitro with little/no impact on regulatory or CD4 T cells. Limited cytokines were detected following 3–4 days culture with human PBMCs. PDL1-ISA had similar potencies for both human and cynomolgus CD8 T cells, and a 2–3-fold lower potency for mouse cells. Pharmacodynamic studies in humanized and BALB/c mice showed transient and dose-dependent increases in circulating NK and CD8 T cells. PDL1-ISA enhanced in vitro killing of PD-L1 positive MDA-MB-231-Luc cells by human PBMCs, CD8 T and NK cells compared to the anti-PD-L1 IgM (no IL-15). PDL1-ISA also demonstrated efficacy in a hPD-L1-CT26 HuCELL mouse model, with most treated animals having complete tumor regressions. Durable anti-tumor immune memory responses were observed upon tumor re-challenge.ConclusionsWe have engineered an IL-15 immunostimulatory anti-PD-L1 IgM antibody that binds PD-L1 more potently than an IgG, stimulates NK and CD8 expansion in vitro and in vivo and induces complete tumor regressions in mouse models. This approach may enhance tumor localization of immunostimulatory cytokine IL-15 though the high affinity and high avidity binding to PD-L1 to improve anti-tumor responses and minimize toxicity.

Expression of Colorectal Cancer Antigenic Protein Fused to IgM Fc in Chinese Cabbage (Brassica rapa)

The epithelial cell adhesion molecule (EpCAM) is a tumor-associated antigen and a potential target for tumor vaccine. The EpCAM is a cell-surface glycoprotein highly expressed in colorectal carcinomas. The objective of the present study is to develop an edible vaccine system through Agrobacterium-mediated transformation in Chinese cabbage (Brassica rapa). For the transformation, two plant expression vectors containing genes encoding for the EpCAM recombinant protein along with the fragment crystallizable (Fc) region of immunoglobulin M (IgM) and Joining (J)-chain tagged with the KDEL endoplasmic reticulum retention motif (J-chain K) were constructed. The vectors were successfully transformed and expressed in the Chinese cabbage individually using Agrobacterium. The transgenic Chinese cabbages were screened using genomic polymerase chain reaction (PCR) in T0 transgenic plant lines generated from both transformants. Similarly, the immunoblot analysis revealed the expression of recombinant proteins in the transformants. Further, the T1 transgenic plants were generated by selfing the transgenic plants (T0) carrying EpCAM–IgM Fc and J-chain K proteins, respectively. Subsequently, the T1 plants generated from EpCAM–IgM Fc and J-chain K transformants were crossed to generate F1 plants carrying both transgenes. The presence of both transgenes was validated using PCR in the F1 plants. In addition, the expression of Chinese cabbage-derived EpCAM–IgM Fc × J-chain K was evaluated using immunoblot and ELISA analyses in the F1 plants. The outcomes of the present study can be utilized for the development of a potential anti-cancer vaccine candidate using Chinese cabbage.

Structure of the Human Secretory Immunoglobulin M Core

Immunoglobulins (Ig) A and M are the only human antibodies that form oligomers and undergo transcytosis to mucosal secretions via the polymeric Ig receptor (pIgR). When complexed with the J-chain (JC) and the secretory component (SC) of pIgR, secretory IgA and IgM (sIgA and sIgM) play critical roles in host-pathogen defense. Recently, we determined the structure of sIgA-Fc which elucidated the mechanism of polymeric IgA assembly and revealed an extensive binding interface between IgA-Fc, JC, and SC. Despite low sequence identity shared with IgA-Fc, IgM-Fc also undergoes JC-mediated assembly and binds pIgR. Here, we report the structure of sIgM-Fc and carryout a systematic comparison to sIgA-Fc. Our structural analysis reveals a remarkably conserved mechanism of JC-templated oligomerization and SC recognition of both IgM and IgA through highly a conserved network of interactions. These studies reveal the structurally conserved features of sIgM and sIgA required for function in mucosal immunity.

Structural insights into secretory immunoglobulin A and its interaction with a pneumococcal adhesin

Secretory Immunoglobulin A (SIgA) is the most abundant antibody at the mucosal surface. SIgA possesses two additional subunits besides IgA: the joining chain (J-chain) and secretory component (SC). SC is the ectodomain of the polymeric immunoglobulin receptor (pIgR), which functions to transport IgA to the mucosa. The underlying mechanism of how the J-chain and pIgR/SC facilitates the assembly and secretion of SIgA remains to be understood. During the infection of Streptococcus pneumoniae, a pneumococcal adhesin SpsA hijacks SIgA and unliganded pIgR/SC to evade host defense and gain entry to human cells. How SpsA specifically targets SIgA and pIgR/SC also remains unclear. Here we report a cryo-electron microscopy structure of the Fc region of human IgA1 (Fcα) in complex with J-chain and SC (Fcα-J-SC), which reveals the organization principle of SIgA. We also present the structure of Fcα-J-SC in complex with SpsA, which uncovers the specific interaction between SpsA and human pIgR/SC. These results advance the molecular understanding of SIgA and shed light on the pathogenesis of S. pneumoniae.

Structural insights into immunoglobulin M

Immunoglobulin M (IgM) plays a pivotal role in both humoral and mucosal immunity. Its assembly and transport depend on the joining chain (J-chain) and the polymeric immunoglobulin receptor (pIgR), but the underlying molecular mechanisms of these processes are unclear. We report a cryo–electron microscopy structure of the Fc region of human IgM in complex with the J-chain and pIgR ectodomain. The IgM-Fc pentamer is formed asymmetrically, resembling a hexagon with a missing triangle. The tailpieces of IgM-Fc pack into an amyloid-like structure to stabilize the pentamer. The J-chain caps the tailpiece assembly and bridges the interaction between IgM-Fc and the polymeric immunoglobulin receptor, which undergoes a large conformational change to engage the IgM-J complex. These results provide a structural basis for the function of IgM.