Oral bacteria induce IgA autoantibodies against a mesangial protein in IgA nephropathy model mice.

IgA nephropathy (IgAN) is caused by deposition of IgA in the glomerular mesangium. The mechanism of selective deposition and production of IgA is unclear; however, we recently identified the involvement of IgA autoantibodies. Here, we show that CBX3 is another self-antigen for IgA in gddY mice, a spontaneous IgAN model, and in IgAN patients. A recombinant antibody derived from gddY mice bound to CBX3 expressed on the mesangial cell surface in vitro and to glomeruli in vivo. An elemental diet and antibiotic treatment decreased the levels of autoantibodies and IgAN symptoms in gddY mice. Serum IgA and the recombinant antibody from gddY mice also bound to oral bacteria of the mice and binding was competed with CBX3. One species of oral bacteria was markedly decreased in elemental diet-fed gddY mice and induced anti-CBX3 antibody in normal mice upon immunization. These data suggest that particular oral bacteria generate immune responses to produce IgA that cross-reacts with mesangial cells to initiate IgAN.

SpiB regulates the expression of B-cell-related genes and increases the longevity of memory B cells.

Generation of memory B cells is one of the key features of adaptive immunity as they respond rapidly to re-exposure to the antigen and generate functional antibodies. Although the functions of memory B cells are becoming clearer, the regulation of memory B cell generation and maintenance is still not well understood. Here we found that transcription factor SpiB is expressed in some germinal center (GC) B cells and memory B cells and participates in the maintenance of memory B cells. Overexpression and knockdown analyses revealed that SpiB suppresses plasma cell differentiation by suppressing the expression of Blimp1 while inducing Bach2 in the in-vitro-induced germinal center B (iGB) cell culture system, and that SpiB facilitates in-vivo appearance of memory-like B cells derived from the iGB cells. Further analysis in IgG1+ cell-specific SpiB conditional knockout (cKO) mice showed that function of SpiB is critical for the generation of late memory B cells but not early memory B cells or GC B cells. Gene expression analysis suggested that SpiB-dependent suppression of plasma cell differentiation is independent of the expression of Bach2. We further revealed that SpiB upregulates anti-apoptosis and autophagy genes to control the survival of memory B cells. These findings indicate the function of SpiB in the generation of long-lasting memory B cells to maintain humoral memory.

A B cell-driven EAE mouse model reveals the impact of B cell-derived cytokines on CNS autoimmunity.

In multiple sclerosis (MS), pathogenic T cell responses are known to be important drivers of autoimmune inflammation. However, increasing evidence suggests an additional role for B cells, which may contribute to pathogenesis via antigen presentation and production of proinflammatory cytokines. However, these B cell effector functions are not featured well in classical experimental autoimmune encephalomyelitis (EAE) mouse models. Here, we compared properties of myelin oligodendrocyte glycoprotein (MOG)-specific and polyclonal B cells and developed an adjuvant-free cotransfer EAE mouse model, where highly activated, MOG-specific induced germinal center B cells provide the critical stimulus for disease development. We could show that high levels of MOG-specific immunoglobulin G (IgGs) are not required for EAE development, suggesting that antigen presentation and activation of cognate T cells by B cells may be important for pathogenesis. As our model allows for B cell manipulation prior to transfer, we found that overexpression of the proinflammatory cytokine interleukin (IL)-6 by MOG-specific B cells leads to an accelerated EAE onset accompanied by activation/expansion of the myeloid compartment rather than a changed T cell response. Accordingly, knocking out IL-6 or tumor necrosis factor α in MOG-specific B cells via CRISPR-Cas9 did not affect activation of pathogenic T cells. In summary, we generated a tool to dissect pathogenic B cell effector function in EAE development, which should improve our understanding of pathogenic processes in MS.

DNASE1L3 enhances antitumor immunity and suppresses tumor progression in colon cancer.

DNASE1L3, an enzyme highly expressed in DCs, is functionally important for regulating autoimmune responses to self-DNA and chromatin. Deficiency of DNASE1L3 leads to development of autoimmune diseases in both humans and mice. However, despite the well-established causal relationship between DNASE1L3 and immunity, little is known about the involvement of DNASE1L3 in regulation of antitumor immunity, the foundation of modern antitumor immunotherapy. In this study, we identify DNASE1L3 as a potentially new regulator of antitumor immunity and a tumor suppressor in colon cancer. In humans, DNASE1L3 is downregulated in tumor-infiltrating DCs, and this downregulation is associated with poor patient prognosis and reduced tumor immune cell infiltration in many cancer types. In mice, Dnase1l3 deficiency in the tumor microenvironment enhances tumor formation and growth in several colon cancer models. Notably, the increased tumor formation and growth in Dnase1l3-deficient mice are associated with impaired antitumor immunity, as evidenced by a substantial reduction of cytotoxic T cells and a unique subset of DCs. Consistently, Dnase1l3-deficient DCs directly modulate cytotoxic T cells in vitro. To our knowledge, our study unveils a previously unknown link between DNASE1L3 and antitumor immunity and further suggests that restoration of DNASE1L3 activity may represent a potential therapeutic approach for anticancer therapy.

Subcutaneous immunisation with zymosan generates mucosal IgA-eliciting memory and protects mice from heterologous influenza virus infection.

Immunoglobulin A (IgA) is the most abundant isotype of antibodies and provides a first line of defense at the mucosa against pathogens invading the host. It has been widely accepted that the mucosal IgA response provided by vaccination requires mucosal inoculation, and intranasal inoculation has been proposed for vaccines against influenza virus. Considering the difficulty of intranasal vaccination in infants or elderly people, however, parenteral vaccination that provides the mucosal IgA response is desirable. Here, we demonstrate that subcutaneous immunisation with zymosan, a yeast cell wall constituent known to be recognised by Dectin-1 and TLR2, potentiates the production of antigen-specific IgA antibodies in the sera and airway mucosa upon intranasal antigen challenge. We confirmed that the antigen-specific IgA-secreting cells accumulated in the lung and nasal-associated lymphoid tissues after the antigen challenge. Such an adjuvant effect of zymosan in the primary immunisation for the IgA response depended on Dectin-1 signalling, but not on TLR2. The IgA response to the antigen challenge required both antigen-specific memory B and T cells, and the generation of memory T cells, but not memory B cells, depended on zymosan as an adjuvant. Finally, we demonstrated that subcutaneous inoculation of inactivated influenza virus with zymosan, but not with alum, mostly protected the mice from infection with a lethal dose of a heterologous virus strain. These data suggest that zymosan is a possible adjuvant for parenteral immunisation that generates memory IgA responses to respiratory viruses such as influenza virus.

Incomplete Atypical Femoral Fracture With Severe Bowing Treated by Intramedullary Nail and Corrective Osteotomy.

We present the case of an 82-year-old female who had difficulty walking due to right thigh pain caused by incomplete atypical femoral fracture (AFF). The femoral bowing was so severe that intramedullary nail insertion was impossible, so we performed a corrective osteotomy of the femur and inserted the intramedullary nail. Postoperatively, the femoral pain disappeared, and bone fusion was achieved at one year and two months postoperatively. In cases of incomplete AFF with very severe femoral bowing, internal fixation with an intramedullary nail combined with corrective osteotomy of the femur is useful.

Identification of IgA autoantibodies targeting mesangial cells redefines the pathogenesis of IgA nephropathy.

Immunoglobulin A (IgA) nephropathy (IgAN) is the most common type of primary glomerulonephritis, often progressing to renal failure. IgAN is triggered by IgA deposition in the glomerular mesangium by an undefined mechanism. Here, we show that grouped ddY (gddY) mice, a spontaneous IgAN model, produce serum IgA against mesangial antigens, including ßII-spectrin. Most patients with IgAN also have serum anti-ßII-spectrin IgA. As in patients with IgAN, IgA+ plasmablasts accumulate in the kidneys of gddY mice. IgA antibodies cloned from the plasmablasts carry substantial V-region mutations and bind to ßII-spectrin and the surface of mesangial cells. These IgAs recognize transfected and endogenous ßII-spectrin exposed on the surface of embryonic kidney-derived cells. Last, we demonstrate that the cloned IgA can bind selectively to glomerular mesangial regions in situ. The identification of IgA autoantibody and its antigen in IgAN provides key insights into disease onset and redefines IgAN as a tissue-specific autoimmune disease.

Commensal Bacteria and the Lung Environment Are Responsible for Th2-Mediated Memory Yielding Natural IgE in MyD88-Deficient Mice.

IgE Abs are a common mediator of allergic responses and are generally produced in type 2 immune responses to allergens. Allergen stimulation of IgE-bound FcεRI on mast cells or basophils induces the production of chemical mediators and cytokines. In addition, IgE binding to FcεRI without allergen promotes the survival or proliferation of these and other cells. Thus, spontaneously produced natural IgE can increase an individual's susceptibility to allergic diseases. Mice deficient in MyD88, a major TLR signaling molecule, have high serum levels of natural IgE, the mechanism for which remains unknown. In this study, we demonstrated that the high serum IgE levels were maintained from weaning by memory B cells (MBCs). IgE from plasma cells and sera from most Myd88-/- mice, but none of the Myd88+/- mice, recognized Streptococcus azizii, a commensal bacterium overrepresented in the lungs of Myd88-/- mice. IgG1+ MBCs from the spleen also recognized S. azizii. The serum IgE levels declined with the administration of antibiotics and were boosted by challenge with S. azizii in Myd88-/- mice, indicating the contribution of S. azizii-specific IgG1+ MBCs to the natural IgE production. Th2 cells were selectively increased in the lungs of Myd88-/- mice and were activated upon addition of S. azizii in the lung cells ex vivo. Finally, lung nonhematopoietic cells, and CSF1 overproduced therefrom, were responsible for natural IgE production in Myd88-/- mice. Thus, some commensal bacteria may prime the Th2 response and natural IgE production in the MyD88-defective lung environment in general.

Upregulated Expression of the IL-9 Receptor on TRAF3-Deficient B Lymphocytes Confers Ig Isotype Switching Responsiveness to IL-9 in the Presence of Antigen Receptor Engagement and IL-4.

The pleiotropic cytokine IL-9 signals to target cells by binding to a heterodimeric receptor consisting of the unique subunit IL-9R and the common subunit γ-chain shared by multiple cytokines of the γ-chain family. In the current study, we found that the expression of IL-9R was strikingly upregulated in mouse naive follicular B cells genetically deficient in TNFR-associated factor 3 (TRAF3), a critical regulator of B cell survival and function. The highly upregulated IL-9R on Traf3-/- follicular B cells conferred responsiveness to IL-9, including IgM production and STAT3 phosphorylation. Interestingly, IL-9 significantly enhanced class switch recombination to IgG1 induced by BCR crosslinking plus IL-4 in Traf3-/- B cells, which was not observed in littermate control B cells. We further demonstrated that blocking the JAK-STAT3 signaling pathway abrogated the enhancing effect of IL-9 on class switch recombination to IgG1 induced by BCR crosslinking plus IL-4 in Traf3-/- B cells. Our study thus revealed, to our knowledge, a novel pathway that TRAF3 suppresses B cell activation and Ig isotype switching by inhibiting IL-9R-JAK-STAT3 signaling. Taken together, our findings provide (to our knowledge) new insights into the TRAF3-IL-9R axis in B cell function and have significant implications for the understanding and treatment of a variety of human diseases involving aberrant B cell activation such as autoimmune disorders.

B cell-intrinsic DNase1L3 is essential for the T cell-independent type II response in mice.

T cell independent type II (TI-II) antigens, such as capsular polysaccharides, have multivalent epitopes, which induce B cell activation, plasma cell differentiation and antibody production by strongly cross-linking B cell receptors. However, the mechanism of B cell activation by TI-II antigens remains unclear. In this study, we demonstrate that DNA endonuclease DNase1L3 (also termed DNase γ) is required for the TI-II response. The production of antigen-specific antibodies was severely diminished in DNase1L3-deficient mice upon immunization with TI-II antigens, but not with T cell dependent (TD) antigens. Bone marrow chimeric mice and B cell transfer experiments revealed that B cell-intrinsic DNase1L3 was required for the TI-II response. DNase1L3-deficient B cells were defective in cell proliferation and plasma cell differentiation in the TI-II response in vivo as well as in vitro, which was not rescued by co-culture with DNase1L3-sufficient B cells in vitro, disproving an involvement of a secretory DNase1L3. In vitro stimulation with TI-II antigen transiently increased expression of DNase1L3 and its translocation into the nucleus. RNA-seq analysis of ex vivo B cells that had responded to TI-II antigen in vivo revealed a marked reduction of Myc-target gene sets in DNase1L3-deficient B cells. Expression of IRF4, a gene that Myc targets, was diminished in the ex vivo DNase1L3-deficient B cells, in which forced expression of IRF4 restored the TI-II response in vivo. These data revealed an unexpected role of DNase1L3 in a missing link between B cell receptor signaling and B cell activation in the TI-II response, giving a valuable clue to molecularly dissect this response.

RelB contributes to the survival, migration and lymphomagenesis of B cells with constitutively active CD40 signaling.

Activation of CD40-signaling contributes to the initiation, progression and drug resistance of B cell lymphomas. We contributed to this knowledge by showing that constitutive CD40-signaling in B cells induces B cell hyperplasia and finally B cell lymphoma development in transgenic mice. CD40 activates, among others, the non-canonical NF-ĸB signaling, which is constitutively activated in several human B cell lymphomas and is therefore presumed to contribute to lymphopathogenesis. This prompted us to study the regulatory role of the non-canonical NF-ĸB transcription factor RelB in lymphomagenesis. To this end, we crossed mice expressing a constitutively active CD40 receptor in B cells with conditional RelB-KO mice. Ablation of RelB attenuated pre-malignant B cell expansion, and resulted in an impaired survival and activation of long-term CD40-stimulated B cells. Furthermore, we found that hyperactivation of non-canonical NF-кB signaling enhances the retention of B cells in the follicles of secondary lymphoid organs. RNA-Seq-analysis revealed that several genes involved in B-cell migration, survival, proliferation and cytokine signaling govern the transcriptional differences modulated by the ablation of RelB in long-term CD40-stimulated B cells. Inactivation of RelB did not abrogate lymphoma development. However, lymphomas occurred with a lower incidence and had a longer latency period. In summary, our data suggest that RelB, although it is not strictly required for malignant transformation, accelerates the lymphomagenesis of long-term CD40-stimulated B cells by regulating genes involved in migration, survival and cytokine signaling.

cis interaction of CD153 with TCR/CD3 is crucial for the pathogenic activation of senescence-associated T cells.

With age, senescence-associated (SA) CD4+ T cells that are refractory to T cell receptor (TCR) stimulation are increased along with spontaneous germinal center (Spt-GC) development prone to autoantibody production. We demonstrate that CD153 and its receptor CD30 are expressed in SA-T and Spt-GC B cells, respectively, and deficiency of either CD153 or CD30 results in the compromised increase of both cell types. CD153 engagement on SA-T cells upon TCR stimulation causes association of CD153 with the TCR/CD3 complex and restores TCR signaling, whereas CD30 engagement on GC B cells induces their expansion. Administration of an anti-CD153 antibody blocking the interaction with CD30 suppresses the increase in both SA-T and Spt-GC B cells with age and ameliorates lupus in lupus-prone mice. These results suggest that the molecular interaction of CD153 and CD30 plays a central role in the reciprocal activation of SA-T and Spt-GC B cells, leading to immunosenescent phenotypes and autoimmunity.

Mouse pulmonary interstitial macrophages mediate the pro-tumorigenic effects of IL-9.

Although IL-9 has potent anti-tumor activity in adoptive cell transfer therapy, some models suggest that it can promote tumor growth. Here, we show that IL-9 signaling is associated with poor outcomes in patients with various forms of lung cancer, and is required for lung tumor growth in multiple mouse models. CD4+ T cell-derived IL-9 promotes the expansion of both CD11c+ and CD11c- interstitial macrophage populations in lung tumor models. Mechanistically, the IL-9/macrophage axis requires arginase 1 (Arg1) to mediate tumor growth. Indeed, adoptive transfer of Arg1+ but not Arg1- lung macrophages to Il9r-/- mice promotes tumor growth. Moreover, targeting IL-9 signaling using macrophage-specific nanoparticles restricts lung tumor growth in mice. Lastly, elevated expression of IL-9R and Arg1 in tumor lesions is associated with poor prognosis in lung cancer patients. Thus, our study suggests the IL-9/macrophage/Arg1 axis is a potential therapeutic target for lung cancer therapy.

An IL-9-pulmonary macrophage axis defines the allergic lung inflammatory environment.

Despite IL-9 functioning as a pleiotropic cytokine in mucosal environments, the IL-9-responsive cell repertoire is still not well defined. Here, we found that IL-9 mediates proallergic activities in the lungs by targeting lung macrophages. IL-9 inhibits alveolar macrophage expansion and promotes recruitment of monocytes that develop into CD11c+ and CD11c- interstitial macrophage populations. Interstitial macrophages were required for IL-9-dependent allergic responses. Mechanistically, IL-9 affected the function of lung macrophages by inducing Arg1 activity. Compared with Arg1-deficient lung macrophages, Arg1-expressing macrophages expressed greater amounts of CCL5. Adoptive transfer of Arg1+ lung macrophages but not Arg1- lung macrophages promoted allergic inflammation that Il9r-/- mice were protected against. In parallel, the elevated expression of IL-9, IL-9R, Arg1, and CCL5 was correlated with disease in patients with asthma. Thus, our study uncovers an IL-9/macrophage/Arg1 axis as a potential therapeutic target for allergic airway inflammation.

Integrin CD11b provides a new marker of pre-germinal center IgA+ B cells in murine Peyer's patches.

Activated B cells can enter germinal centers (GCs) for affinity maturation to produce high-affinity antibodies. However, which activated B cells will enter GCs remains unknown. Here, we found a small population of CD11b+IgA+ B cells located outside of GCs in murine Peyer's patches (PPs). After injection of the CD11b+IgA+ PP B cells into a PP of a recipient mouse, they entered GCs forty hours later. They expressed GC surface markers and pre-GC B cell genes, suggesting that CD11b provides a novel surface marker of pre-GC IgA+ B cells in murine PPs. Furthermore, independently of dendritic cell activation, CD11b expression on B cells can be induced by bacterial antigens, such as pam3CSK4 and heat-killed Escherichia coli in vitro. In addition, mice orally administered with pam3CSK4 or heat-killed E. coli increased the number of PP GC B cells within two days, and enhanced the mucosal antigen-specific IgA response. Our results demonstrate that the induction of CD11b on B cells is a promising marker for selecting an effective mucosal vaccine adjuvant.

C/EBPß induces B-cell acute lymphoblastic leukemia and cooperates with BLNK mutations.

BLNK (BASH/SLP-65) encodes an adaptor protein that plays an important role in B-cell receptor (BCR) signaling. Loss-of-function mutations in this gene are observed in human pre-B acute lymphoblastic leukemia (ALL), and a subset of Blnk knock-out (KO) mice develop pre-B-ALL. To understand the molecular mechanism of the Blnk mutation-associated pre-B-ALL development, retroviral tagging was applied to KO mice using the Moloney murine leukemia virus (MoMLV). The Blnk mutation that significantly accelerated the onset of MoMLV-induced leukemia and increased the incidence of pre-B-ALL Cebpb was identified as a frequent site of retroviral integration, suggesting that its upregulation cooperates with Blnk mutations. Transgenic expression of the liver-enriched activator protein (LAP) isoform of Cebpb reduced the number of mature B-lymphocytes in the bone marrow and inhibited differentiation at the pre-BI stage. Furthermore, LAP expression significantly accelerated leukemogenesis in Blnk KO mice and alone acted as a B-cell oncogene. Furthermore, an inverse relationship between BLNK and C/EBPß expression was also noted in human pre-B-ALL cases, and the high level of CEBPB expression was associated with short survival periods in patients with BLNK-downregulated pre-B-ALL. These results indicate the association between the C/EBPß transcriptional network and BCR signaling in pre-B-ALL development and leukemogenesis. This study gives insight into ALL progression and suggests that the BCR/C/EBPß pathway can be a therapeutic target.

Protein kinase Cδ is essential for the IgG response against T-cell-independent type 2 antigens and commensal bacteria.

Antigens (Ags) with multivalent and repetitive structure elicit IgG production in a T-cell-independent manner. However, the mechanisms by which such T-cell-independent type-2 (TI-2) Ags induce IgG responses remain obscure. Here, we report that B-cell receptor (BCR) engagement with a TI-2 Ag but not with a T-cell-dependent (TD) Ag was able to induce the transcription of Aicda encoding activation-induced cytidine deaminase (AID) and efficient class switching to IgG3 upon costimulation with IL-1 or IFN-α in mouse B cells. TI-2 Ags strongly induced the phosphorylation of protein kinase C (PKC)δ and PKCδ mediated the Aicda transcription through the induction of BATF, the key transcriptional regulator of Aicda. In PKCδ-deficient mice, production of IgG was intact against TD Ag but abrogated against typical TI-2 Ags as well as commensal bacteria, and experimental disruption of the gut epithelial barrier resulted in fatal bacteremia. Thus, our results have revealed novel molecular requirements for class switching in the TI-2 response and highlighted its importance in homeostatic commensal-specific IgG production.

When the human body faces a potentially harmful microorganism, the immune system responds by finding and destroying the pathogen. This involves the coordination of several different parts of the immune system. B cells are a type of white blood cell that is responsible for producing antibodies: large proteins that bind to specific targets such as pathogens. B cells often need help from other immune cells known as T cells to complete antibody production. However, T cells are not required for B cells to produce antibodies against some bacteria. For example, when certain pathogenic bacteria coated with a carbohydrate called a capsule ­ such as pneumococcus, which causes pneumonia, or salmonella ­ invade our body, B cells recognize a repetitive structure of the capsule using a B-cell antigen receptor. This recognition allows B cells to produce antibodies independently of T cells. It is unclear how B cells produce antibodies in this situation or what proteins are required for this activity. To understand this process, Fukao et al. used genetically modified mice and their B cells to study how they produce antibodies independently of T cells. They found that a protein called PKCδ is critical for B cells to produce antibodies, especially of an executive type called IgG, in the T-cell-independent response. PKCδ became active when B cells were stimulated with the repetitive antigen present on the surface of bacteria like salmonella or pneumococcus. Mice that lack PKCδ were unable to produce IgG independently of T cells, leading to fatal infections when bacteria reached the tissues and blood. Understanding the mechanism behind the T cell-independent B cell response could lead to more effective antibody production, potentially paving the way for new vaccines to prevent fatal diseases caused by pathogenic bacteria.

MicroRNA-directed pathway discovery elucidates an miR-221/222-mediated regulatory circuit in class switch recombination.

MicroRNAs (miRNAs, miRs) regulate cell fate decisions by post-transcriptionally tuning networks of mRNA targets. We used miRNA-directed pathway discovery to reveal a regulatory circuit that influences Ig class switch recombination (CSR). We developed a system to deplete mature, activated B cells of miRNAs, and performed a rescue screen that identified the miR-221/222 family as a positive regulator of CSR. Endogenous miR-221/222 regulated B cell CSR to IgE and IgG1 in vitro, and miR-221/222-deficient mice exhibited defective IgE production in allergic airway challenge and polyclonal B cell activation models in vivo. We combined comparative Ago2-HITS-CLIP and gene expression analyses to identify mRNAs bound and regulated by miR-221/222 in primary B cells. Interrogation of these putative direct targets uncovered functionally relevant downstream genes. Genetic depletion or pharmacological inhibition of Foxp1 and Arid1a confirmed their roles as key modulators of CSR to IgE and IgG1.

Mechanisms for the regulation of memory B-cell recall responses in mice.

Upon infection by pathogens or vaccination, the adaptive immune system rapidly but transiently produces antibodies. Some weeks later, however, long-lasting immunity is established that protects the host against the same pathogens almost for life through continuous production of antibodies on one hand and the maintenance of cytotoxic T cells on the other, collectively called immunological memory. The antibody-mediated arm, also called serological memory, is mainly exerted by long-lived plasma cells and memory B cells (MBCs). MBCs express receptors for the specific pathogens and circulate to survey the body for almost a life-long period. Upon recognizing the pathogen, MBCs clonally expand and produce a large amount of the specific antibodies to stop the infection, the process called a (memory) recall response. Although such a function of MBCs has long been known, the mechanism of how their performance is regulated has been obscure. This is due to their paucity in the body, lack of definitive surface markers and obscure ontogeny. However, recent studies have revealed the multifold mechanisms by which the recall response of MBCs is regulated: rapid and enhanced antibody production is due to a mechanism intrinsic to MBCs, namely, up-regulated expression levels of surface molecules interacting with T cells and the property of IgG-class antigen receptors; to a property of the responsible subset of MBCs; and to co-stimulation through innate receptors and cytokines. It has also been unveiled that the recall response is negatively regulated by an inhibitory receptor on MBCs and by antigens with repetitive epitopes.

A novel cancer immunotherapy using tumor-infiltrating B cells in the APCmin/+ mouse model.

Accumulating evidence has suggested a correlation of tumor infiltrating B cells (TiBcs) and a good prognosis of cancer diseases. In some cases, TiBcs appear to have experienced antigen stimulation since they have undergone class-switching and somatic hypermutation and formed tertiary lymphoid structures around tumors together with T cells. Assuming TiBcs include those that recognize some tumor antigens, we sought to investigate their possible usefulness for cell-mediated immunotherapies. To expand usually small number of TiBcs in vitro, we modified our B cell culture system: we transduced B cells with ERT2-Bach2 so that they grow unlimitedly provided with tamoxifen, IL-21 and our original feeder cells. Such cells differentiate into plasma cells and produce antibodies upon withdrawal of tamoxifen, and further by addition of a Bach2-inhibitor in vitro. As a preliminary experiment, thus expanded splenic B cells expressing a transgenic antigen receptor/antibody against hen egg lysozyme were intravenously injected into mice pre-implanted with B16 melanoma cells expressing membrane-bound HEL in the skin, which resulted in suppression of the growth of B16 tumors and prolonged survival of the recipient mice. To test the usefulness of TiBcs for the immunotherapy, we next used APCmin/+ mice as a model that spontaneously develop intestinal tumors. We cultured TiBcs separated from the tumors of APCmin/+ mice as above and confirmed that the antibodies they produce recognize the APCmin/+ tumor. Repeated injection of such TiBcs into adult APCmin/+ mice resulted in suppression of intestinal tumor growth and elongation of the survival of the recipient mice. Serum antibody from the TiBc-recipient mice selectively bound to an antigen expressed in the tumor of APCmin/+ mice. These data suggest a possibility of the novel individualized cancer immunotherapy, in which TiBcs from surgically excised tumor tissues are expanded and infused into the donor patients.