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Newly generated immature B cells that bind self-antigen with high avidity arrest in differentiation and undergo central tolerance via receptor editing and clonal deletion. These autoreactive immature B cells also express low surface levels of the coreceptor CD19, a key activator of the PI3K pathway. Signals emanating from both CD19 and PI3K are known to be critical for attenuating receptor editing and selecting immature B cells into the periphery. However, the mechanisms that modulate CD19 expression at this stage of B cell development have not yet been resolved. Using in vivo and in vitro models, we demonstrate that Cd19 de novo gene transcription and translation do not significantly contribute to the differences in CD19 surface expression in mouse autoreactive and nonautoreactive immature B cells. Instead, CD19 downregulation is induced by BCR stimulation in proportion to BCR engagement, and the remaining surface IgM and CD19 molecules promote intracellular PI3K-AKT activity in proportion to their level of expression. The internalized CD19 is degraded with IgM by the lysosome, but inhibiting lysosome-mediated protein degradation only slightly improves surface CD19. In fact, CD19 is restored only upon Ag removal. Our data also reveal that the PI3K-AKT pathway positively modulates CD19 surface expression in immature B cells via a mechanism that is independent of inhibition of FOXO1 and its role on Cd19 gene transcription while is dependent on mTORC1.

CD8 T lymphocytes are able to eliminate nascent tumor cells through a process referred to as immunosurveillance. However, multiple inhibitory mechanisms within the tumor microenvironment have been described that impede tumor rejection by CD8 T cells, including increased signaling by inhibitory receptors. Lysophosphatidic acid (LPA) is a bioactive lysophospholipid that has been shown repeatedly to promote diverse cellular processes benefiting tumorigenesis. Accordingly, the exaggerated expression of LPA and LPA receptors is a common feature of diverse tumor cell lineages and can result in elevated systemic LPA levels. LPA is recognized by at least six distinct G protein–coupled receptors, several of which are expressed by T cells, although the precise function of LPA signaling in CD8 T-cell activation and function has not been defined. Here, we show that LPA signaling via the LPA5 receptor expressed by CD8 T cells suppresses antigen receptor signaling, cell activation, and proliferation in vitro and in vivo. Importantly, in a mouse melanoma model tumor-specific CD8 T cells that are LPA5-deficient are able to control tumor growth significantly better than wild-type tumor-specific CD8 T cells. Together, these data suggest that the production of LPA by tumors serves not only in an autocrine manner to promote tumorigenesis, but also as a mechanism to suppress adaptive immunity and highlights a potential novel target for cancer treatment. Cancer Immunol Res; 1(4); 245–55. ©2013 AACR.

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The random recombination of immunoglobulin V(D)J gene segments produces unique IgM antibodies that serve as the antigen receptor for each developing B cell. Hence, the newly formed B cell repertoire is comprised of a variety of specificities that display a range of reactivity with self-antigens. Newly generated IgM(+) immature B cells that are non-autoreactive or that bind self-antigen with low avidity are licensed to leave the bone marrow with their intact antigen receptor and to travel via the blood to the peripheral lymphoid tissue for further selection and maturation. In contrast, clones with medium to high avidity for self-antigen remain within the marrow and undergo central tolerance, a process that revises their antigen receptor or eliminates the autoreactive B cell altogether. Thus, central B cell tolerance is critical for reducing the autoreactive capacity and avidity for self-antigen of our circulating B cell repertoire. Bone marrow cultures and mouse models have been instrumental for understanding the mechanisms that regulate the selection of bone marrow B cells. Here we review recent studies that have shed new light on the contribution of the ERK, PI3K, and CXCR4 signaling pathways in the selection of mouse and human immature B cells that either bind or do not bind self-antigen.

Reversing the immunosuppressive nature of the tumor microenvironment is critical for the successful treatment of cancers with immunotherapy drugs. Murine cancer models are extremely limited in their diversity and suffer from poor translation to the clinic. To serve as a more physiological preclinical model for immunotherapy studies, this protocol has been developed to evaluate the treatment of human tumors in a mouse reconstituted with a human immune system. This unique protocol demonstrates the development of human immune system (HIS, "humanized") mice, followed by implantation of a human tumor, either a cell-line derived xenograft (CDX) or a patient derived xenograft (PDX). HIS mice are generated by injecting CD34+ human hematopoietic stem cells isolated from umbilical cord blood into neonatal BRGS (BALB/c Rag2

Lysophosphatidic acid (LPA) is a bioactive lipid which increases in concentration locally and systemically across different cancer types. Yet, the exact mechanism(s) of how LPA affects CD8 T cell immunosurveillance during tumor progression remain unknown. We show LPA receptor (LPAR) signaling by CD8 T cells promotes tolerogenic states via metabolic reprogramming and potentiating exhaustive differentiation to modulate anti-tumor immunity. We found LPA levels predict response to immunotherapy and Lpar5 signaling drives exhausted phenotypes on CD8 T cells. Importantly, we identify a novel function of Lpar5 to regulate CD8 T cell respiration, proton leak, and reactive oxygen species. Together, our findings reveal that LPA serves as a lipid-regulated immune checkpoint by modulating metabolic efficiency through LPAR5 signaling on CD8 T cells. Our study offers key insights into the mechanisms governing adaptive anti-tumor immunity and demonstrates LPA could be exploited as a novel T cell directed therapy to improve dysfunctional anti-tumor immunity.

Severe SARS-CoV-2 infection is associated with strong inflammation and autoantibody production against diverse self-antigens, suggesting a system-wide defect in B cell tolerance. BND cells are a B cell subset in healthy individuals harboring autoreactive but anergic B lymphocytes. In vitro evidence suggests inflammatory stimuli can breach peripheral B cell tolerance in this subset. We asked whether SARS-CoV-2–associated inflammation impairs BND cell peripheral tolerance. To address this, PBMCs and plasma were collected from healthy controls, individuals immunized against SARS-CoV-2, or subjects with convalescent or severe SARS-CoV-2 infection. We demonstrate that BND cells from severely infected individuals are significantly activated, display reduced inhibitory receptor expression, and restored BCR signaling, indicative of a breach in anergy during viral infection, supported by increased levels of autoreactive antibodies. The phenotypic and functional BND cell alterations significantly correlate with increased inflammation in severe SARS-CoV-2 infection. Thus, autoreactive BND cells are released from peripheral tolerance with SARS-CoV-2 infection, likely as a consequence of robust systemic inflammation.

Cancer and chronic infections often increase levels of the bioactive lipid, lysophosphatidic acid (LPA), that we have demonstrated acts as an inhibitory ligand upon binding LPAR5 on CD8 T cells, suppressing cytotoxic activity and tumor control. This study, using human and mouse primary T lymphocytes, reveals how LPA disrupts antigen-specific CD8 T cell:target cell immune synapse (IS) formation and T cell function via competing for cytoskeletal regulation. Specifically, we find upon antigen-specific T cell:target cell formation, IP3R1 localizes to the IS by a process dependent on mDia1 and actin and microtubule polymerization. LPA not only inhibited IP3R1 from reaching the IS but also altered T cell receptor (TCR)–induced localization of RhoA and mDia1 impairing F-actin accumulation and altering the tubulin code. Consequently, LPA impeded calcium store release and IS-directed cytokine secretion. Thus, targeting LPA signaling in chronic inflammatory conditions may rescue T cell function and promote antiviral and antitumor immunity.

Central B cell tolerance, the process restricting the development of many newly generated autoreactive B cells, has been intensely investigated in mouse cells while studies in humans have been hampered by the inability to phenotypically distinguish autoreactive and nonautoreactive immature B cell clones and the difficulty in accessing fresh human bone marrow samples. Using a human immune system mouse model in which all human Igκ+ B cells undergo central tolerance, we discovered that human autoreactive immature B cells exhibit a distinctive phenotype that includes lower activation of ERK and differential expression of CD69, CD81, CXCR4, and other glycoproteins. Human B cells exhibiting these characteristics were observed in fresh human bone marrow tissue biopsy specimens, although differences in marker expression were smaller than in the humanized mouse model. Furthermore, the expression of these markers was slightly altered in autoreactive B cells of humanized mice engrafted with some human immune systems genetically predisposed to autoimmunity. Finally, by treating mice and human immune system mice with a pharmacologic antagonist, we show that signaling by CXCR4 is necessary to prevent both human and mouse autoreactive B cell clones from egressing the bone marrow, indicating that CXCR4 functionally contributes to central B cell tolerance. This work was supported by the NIH (Grants AI124474 and AI131639 to R.P. and Grant AI136534 to R.M.T.). It was also supported in part by NIH National Center for Advancing Translational Sciences Colorado Clinical and Translational Science Awards Grant UL1 TR002535. The contents are the authors’sole responsibility anddo not necessarily represent official NIH views. Sí

Despite the role of donor-specific antibodies (DSAs) in recognizing major histocompatibility complex (MHC) antigens and mediating transplant rejection, how and where recipient B cells in lymphoid tissues encounter donor MHC antigens remains unclear. Contrary to the dogma, we demonstrated here that migration of donor leukocytes out of skin or heart allografts is not necessary for B or T cell allosensitization in mice. We found that mouse skin and cardiac allografts and human skin grafts release cell-free donor MHC antigens via extracellular vesicles (EVs) that are captured by subcapsular sinus (SCS) macrophages in lymph nodes or analog macrophages in the spleen. Donor EVs were transported across the SCS macrophages, and donor MHC molecules on the EVs were recognized by alloreactive B cells. This triggered B cell activation and DSA production, which were both prevented by SCS macrophage depletion. These results reveal an unexpected role for graft-derived EVs and open venues to interfere with EV biogenesis, trafficking, or function to restrain priming or reactivation of alloreactive B cells.

Background Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited systemic treatment options. RX-5902 is a novel anti-cancer agent that inhibits phosphorylated-p68 and thus attenuates nuclear β-catenin signaling. The purpose of this study was to evaluate the ability of β-catenin signaling blockade to enhance the efficacy of anti-CTLA-4 and anti-PD-1 immune checkpoint blockade in immunocompetent, preclinical models of TNBC. Methods Treatment with RX-5902, anti-PD-1, anti-CTLA-4 or the combination was investigated in BALB/c mice injected with the 4 T1 TNBC cell line. Humanized BALB/c-Rag2nullIl2rγnullSIRPαNOD (hu-CB-BRGS) mice transplanted with a human immune system were implanted with MDA-MB-231 cells. Mice were randomized into treatment groups according to human hematopoietic chimerism and treated with RX-5902, anti-PD-1 or the combination. At sacrifice, bone marrow, lymph nodes, spleen and tumors were harvested for flow cytometry analysis of human immune cells. Results The addition of RX-5902 to CTLA-4 or PD-1 inhibitors resulted in decreased tumor growth in the 4 T1 and human immune system and MDA-MB-231 xenograft models. Immunologic analyses demonstrated a significant increase in the number of activated T cells in tumor infiltrating lymphocytes (TILs) with RX-5902 treatment compared to vehicle (p p Conclusions Conclusions: RX-5902 enhanced the efficacy of nivolumab in a humanized, preclinical model of TNBC. Several changes in immunologic profiles were noted in mice treated with RX-5902 and the combination, including an increase in activated TILs and a decrease in human myeloid populations, that are often associated with immunosuppression in a tumor microenvironment. RX-5902 also was shown to potentiate the effects of checkpoint inhibitors of CTLA4 and the PD-1 inhibitor in the 4 T-1 murine TNBC model. These findings indicate that RX-5902 may have important immunomodulatory, as well as anti-tumor activity, in TNBC when combined with a checkpoint inhibitor.

Peripheral tolerance is essential for silencing weakly autoreactive B cells that have escaped central tolerance, but it is unclear why these potentially pathogenic B cells are retained rather than being eliminated entirely. Release from peripheral tolerance restraint can occur under certain circumstances (i.e., strong TLR stimulus), that are present during infection. In this regard, we hypothesized that autoreactive B cells could function as a reserve population that can be activated to contribute to the humoral immune response, particularly with pathogens, such as HIV-1, that exploit immune tolerance to avoid host defense. In this study, we identify a population of autoreactive B cells with the potential to neutralize HIV-1 and experimentally release them from the functional restrictions of peripheral tolerance. We have previously identified murine monoclonal antibodies that displayed autoreactivity against histone H2A and neutralized HIV-1 in vitro. Here, we identify additional H2A-reactive IgM monoclonal antibodies and demonstrate that they are both autoreactive and polyreactive with self and foreign antigens and are able to neutralize multiple clades of tier 2 HIV-1. Flow cytometric analysis of H2A-reactive B cells in naïve wildtype mice revealed that these B cells are present in peripheral B cell populations and we further document that murine H2A-reactive B cells are restrained by peripheral tolerance mechanisms. Specifically, we show endogenous H2A-reactive B cells display increased expression of the inhibitory mediators CD5 and phosphatase and tensin homolog (PTEN) phosphatase and fail to mobilize calcium upon immunoreceptor stimulation; all characterized markers of anergy. Moreover, we show that toll-like receptor stimulation or provision of CD4 T cell help induces the in vitro production of H2A-reactive antibodies, breaking tolerance. Thus, we have identified a novel poly/autoreactive B cell population that has the potential to neutralize HIV-1 but is silenced by immune tolerance.

Autoreactive B cells have a major function in autoimmunity. A small subset of B cells expressing two distinct B-cell-antigen-receptors (B2R cells) is elevated in many patients with systematic lupus erythematosus (SLE) and in the MRL(/lpr) mouse model of lupus, and is often autoreactive. Here we show, using RNAseq and in vitro and in vivo analyses, signals that are required for promoting B2R cell numbers and effector function in autoimmune mice. Compared with conventional B cells, B2R cells are more responsive to Toll-like receptor 7/9 and type I/II interferon treatment, display higher levels of MHCII and co-receptors, and depend on IL-21 for their homeostasis; moreover they expand better upon T cell-dependent antigen stimulation, and mount a more robust memory response, which are characteristics essential for enhanced (auto)immune responses. Our findings thus provide insights on the stimuli for the expansion of an autoreactive B cell subset that may contribute to the etiology of SLE., Conventional B cells express clonally specific antigen receptors, but a small subset of B cells from patients and mice with systematic lupus erythematosus simultaneously expresses two distinct antigen receptors. Here the authors show that these dual-specificity B cells have higher levels of MHC-II, depend on IL-21 for expansion, and mount stronger memory response.

Schroeder et al. demonstrate that when peripheral tolerance is relaxed, tier 2 HIV-1–neutralizing antibodies can be elicited and identify new autoreactive antibody specificities against histone H2A capable of neutralizing tier 2 HIV-1., A subset of characterized HIV-1 broadly neutralizing antibodies (bnAbs) are polyreactive with additional specificities for self-antigens and it has been proposed immunological tolerance may present a barrier to their participation in protective humoral immunity. We address this hypothesis by immunizing autoimmune-prone mice with HIV-1 Envelope (Env) and characterizing the primary antibody response for HIV-1 neutralization. We find autoimmune mice generate neutralizing antibody responses to tier 2 HIV-1 strains with alum treatment alone in the absence of Env. Importantly, experimentally breaching immunological tolerance in wild-type mice also leads to the production of tier 2 HIV-1–neutralizing antibodies, which increase in breadth and potency following Env immunization. In both genetically prone and experimentally induced mouse models of autoimmunity, increased serum levels of IgM anti-histone H2A autoantibodies significantly correlated with tier 2 HIV-1 neutralization, and anti-H2A antibody clones were found to neutralize HIV-1. These data demonstrate that breaching peripheral tolerance permits a cross-reactive HIV-1 autoantibody response able to neutralize HIV-1.

Autoimmune diseases evolve from complex interactions between the immune system and self-antigens and involve several genetic attributes, environmental triggers and diverse cell types. Research using experimental mouse models has contributed key knowledge on the mechanisms that underlie these diseases in humans, but differences between the mouse and human immune systems can and, at times, do undermine the translational significance of these findings. The use of human immune system (HIS) mice enables the utility of mouse models with greater relevance for human diseases. As the name conveys, these mice are reconstituted with mature human immune cells transferred directly from peripheral blood or via transplantation of human hematopoietic stem cells that nucleate the generation of a complex human immune system. The function of the human immune system in HIS mice has improved over the years with the stepwise development of better models. HIS mice exhibit key benefits of the murine animal model, such as small size, robust and rapid reproduction and ease of experimental manipulation. Importantly, HIS mice also provide an applicable in vivo setting that permit the investigation of the physiological and pathological functions of the human immune system and its response to novel treatments. With the gaining popularity of HIS mice in the last decade, the potential of this model has been exploited for research in basic science, infectious diseases, cancer, and autoimmunity. In this review we focus on the use of HIS mice in autoimmune studies to stimulate further development of these valuable models., Highlights • Human immune system (HIS) mice bear components of the human immune system. • HIS mice engraft with human blood or hematopoietic stem cells, and sometimes thymus. • HIS mice are used to investigate development and function of the human immune system. • Immunological tolerance and autoimmune responses can be studied in HIS mice. • HIS models of autoimmunity vary in complexity and in ability to represent disease.

Over 30 key leaders in the field participated in a 1-day workshop entitled ‘Recent Advances and Opportunities in the Development and Use of Humanized Immune System Mouse Models’ to discuss the benefits and limitations of using human fetal tissue versus non-fetal tissue sources to generate mice with a humanized immune system. This Comment summarizes the workshop discussions, including highlights of some of the key advances made through the use of humanized mice in improving the understanding of immune system function and developing novel therapeutics for the treatment of infectious, immunological and allergic diseases, as well as current challenges in the production, characterization and utilization of these animal models.

Persistent T cell antigen receptor (TCR) signaling by CD8 T cells is a feature of cancer and chronic infections and results in the sustained expression of, and signaling by, inhibitory receptors, which ultimately impair cytotoxic activity via poorly characterized mechanisms. We have previously determined that the LPA5 GPCR expressed by CD8 T cells, upon engaging the lysophosphatidic acid (LPA) bioactive serum lipid, functions as an inhibitory receptor able to negatively regulate TCR signaling. Notably, the levels of LPA and autotaxin (ATX), the phospholipase D enzyme that produces LPA, are often increased in chronic inflammatory disorders such as chronic infections, autoimmune diseases, obesity, and cancer. In this report, we demonstrate that LPA engagement selectively by LPA5 on human and mouse CD8 T cells leads to the inhibition of several early TCR signaling events including intracellular calcium mobilization and ERK activation. We further show that, as a consequence of LPA5 suppression of TCR signaling, the exocytosis of perforin-containing granules is significantly impaired and reflected by repressed in vitro and in vivo CD8 T cell cytolytic activity. Thus, these data not only document LPA5 as a novel inhibitory receptor but also determine the molecular and biochemical mechanisms by which a naturally occurring serum lipid that is elevated under settings of chronic inflammation signals to suppress CD8 T cell killing activity in both human and murine cells. As diverse tumors have repeatedly been shown to aberrantly produce LPA that acts in an autocrine manner to promote tumorigenesis, our findings further implicate LPA in activating a novel inhibitory receptor whose signaling may be therapeutically silenced to promote CD8 T cell immunity.

Persistent T cell antigen receptor (TCR) signaling by CD8 T cells is a feature of cancer and chronic infections and results in the sustained expression of, and signaling by, inhibitory receptors, which ultimately impair cytotoxic activity via poorly characterized mechanisms. We have previously determined that the LPA5 GPCR expressed by CD8 T cells, upon engaging the lysophosphatidic acid (LPA) bioactive serum lipid, functions as an inhibitory receptor able to negatively regulate TCR signaling. Notably, the levels of LPA and autotaxin (ATX), the phospholipase D enzyme that produces LPA, are often increased in chronic inflammatory disorders such as chronic infections, autoimmune diseases, obesity, and cancer. In this report, we demonstrate that LPA engagement selectively by LPA5 on human and mouse CD8 T cells leads to the inhibition of several early TCR signaling events including intracellular calcium mobilization and ERK activation. We further show that, as a consequence of LPA5 suppression of TCR signaling, the exocytosis of perforin-containing granules is significantly impaired and reflected by repressed in vitro and in vivo CD8 T cell cytolytic activity. Thus, these data not only document LPA5 as a novel inhibitory receptor but also determine the molecular and biochemical mechanisms by which a naturally occurring serum lipid that is elevated under settings of chronic inflammation signals to suppress CD8 T cell killing activity in both human and murine cells. As diverse tumors have repeatedly been shown to aberrantly produce LPA that acts in an autocrine manner to promote tumorigenesis, our findings further implicate LPA in activating a novel inhibitory receptor whose signaling may be therapeutically silenced to promote CD8 T cell immunity.

Central B cell tolerance, the process responsible for mitigating the development of autoreactive B cells, has been extensively studied in mice, while little is known of how human B cells are tolerized in the bone marrow due to the inability to phenotypically distinguish autoreactive and non-autoreactive immature B cells and the difficulty in accessing fresh human bone marrow biopsies. Using a human immune system mouse model where all human Igk+ B cells are autoreactive and therefore tolerized in the bone marrow, we show that human autoreactive immature B cells are phenotypically different from non-autoreactive due to the differential expression of CD69, CD81, CXCR4 and other surface glycoproteins, while also exhibiting lower activation of ERK. Upon treatment of human immune system mice and autoreactive mice with a CXCR4 antagonist we show that signaling through this receptor is necessary to prevent both human and mouse autoreactive B cell clones to egress the bone marrow, indicating that CXCR4 functionally contributes to central B cell tolerance.

Lang et al. show in a humanized mouse model that human B cells undergo central tolerance via a combination of receptor editing and clonal deletion., The mechanisms by which B cells undergo tolerance, such as receptor editing, clonal deletion, and anergy, have been established in mice. However, corroborating these mechanisms in humans remains challenging. To study how autoreactive human B cells undergo tolerance, we developed a novel humanized mouse model. Mice expressing an anti–human Igκ membrane protein to serve as a ubiquitous neo self-antigen (Ag) were transplanted with a human immune system. By following the fate of self-reactive human κ+ B cells relative to nonautoreactive λ+ cells, we show that tolerance of human B cells occurs at the first site of self-Ag encounter, the bone marrow, via a combination of receptor editing and clonal deletion. Moreover, the amount of available self-Ag and the genetics of the cord blood donor dictate the levels of central tolerance and autoreactive B cells in the periphery. Thus, this model can be useful for studying specific mechanisms of human B cell tolerance and to reveal differences in the extent of this process among human populations.

Epstein-Barr virus (EBV) has been classified into two strains, EBV type 1 (EBV-1) and EBV type 2 (EBV-2) based on genetic variances and differences in transforming capacity. EBV-1 readily transforms B cells in culture while EBV-2 is poorly transforming. The differing abilities to immortalize B cells in vitro suggest that in vivo these viruses likely use alternative approaches to establish latency. Indeed, we recently reported that EBV-2 has a unique cell tropism for T cells, infecting T cells in culture and in healthy Kenyan infants, strongly suggesting that EBV-2 infection of T cells is a natural part of the EBV-2 life cycle. However, limitations of human studies hamper further investigation into how EBV-2 utilizes T cells. Therefore, BALB/c Rag2(null) IL2rγ(null) SIRPα humanized mice were utilized to develop an EBV-2 in vivo model. Infection of humanized mice with EBV-2 led to infection of both T and B cells, unlike infection with EBV-1, in which only B cells were infected. Gene expression analysis demonstrated that EBV-2 established a latency III infection with evidence of ongoing viral reactivation in both B and T cells. Importantly, EBV-2-infected mice developed tumors resembling diffuse large B cell lymphoma (DLBCL). These lymphomas had morphological features comparable to those of EBV-1-induced DLBCLs, developed at similar rates with equivalent frequencies, and expressed a latency III gene profile. Thus, despite the impaired ability of EBV-2 to immortalize B cells in vitro, EBV-2 efficiently induces lymphomagenesis in humanized mice. Further research utilizing this model will enhance our understanding of EBV-2 biology, the consequence of EBV infection of T cells, and the capacity of EBV-2 to drive lymphomagenesis. IMPORTANCE EBV is a well-established B cell-tropic virus. However, we have recently shown that the EBV type 2 (EBV-2) strain also infects primary T cells in culture and in healthy Kenyan children. This finding suggests that EBV-2, unlike the well-studied EBV-1 strain, utilizes the T cell compartment to persist. As EBV is human specific, studies to understand the role of T cells in EBV-2 persistence require an in vivo model. Thus, we developed an EBV-2 humanized mouse model, utilizing immunodeficient mice engrafted with human cord blood CD34(+) stem cells. Characterization of the EBV-2-infected humanized mice established that both T cells and B cells are infected by EBV-2 and that the majority of infected mice develop a B cell lymphoma resembling diffuse large B cell lymphoma. This new in vivo model can be utilized for studies to enhance our understanding of how EBV-2 infection of T cells contributes to persistence and lymphomagenesis.

Background The success of agents that reverse T-cell inhibitory signals, such as anti-PD-1/PD-L1 therapies, has reinvigorated cancer immunotherapy research. However, since only a minority of patients respond to single-agent therapies, methods to test the potential anti-tumor activity of rational combination therapies are still needed. Conventional murine xenograft models have been hampered by their immune-compromised status; thus, we developed a hematopoietic humanized mouse model, hu-CB-BRGS, and used it to study anti-tumor human immune responses to triple-negative breast cancer (TNBC) cell line and patient-derived colorectal cancer (CRC) xenografts (PDX). Methods BALB/c-Rag2nullIl2rγnullSIRPαNOD (BRGS) pups were humanized through transplantation of cord blood (CB)-derived CD34+ cells. Mice were evaluated for human chimerism in the blood and assigned into experimental untreated or nivolumab groups based on chimerism. TNBC cell lines or tumor tissue from established CRC PDX models were implanted into both flanks of humanized mice and treatments ensued once tumors reached a volume of ~150mm3. Tumors were measured twice weekly. At end of study, immune organs and tumors were collected for immunological assessment. Results Humanized PDX models were successfully established with a high frequency of tumor engraftment. Humanized mice treated with anti-PD-1 exhibited increased anti-tumor human T-cell responses coupled with decreased Treg and myeloid populations that correlated with tumor growth inhibition. Combination therapies with anti-PD-1 treatment in TNBC-bearing mice reduced tumor growth in multi-drug cohorts. Finally, as observed in human colorectal patients, anti-PD-1 therapy had a strong response to a microsatellite-high CRC PDX that correlated with a higher number of human CD8+ IFNγ+ T cells in the tumor. Conclusion Hu-CB-BRGS mice represent an in vivo model to study immune checkpoint blockade to human tumors. The human immune system in the mice is inherently suppressed, similar to a tumor microenvironment, and thus allows growth of human tumors. However, the suppression can be released by anti-PD-1 therapies and inhibit tumor growth of some tumors. The model offers ample access to lymph and tumor cells for in-depth immunological analysis. The tumor growth inhibition correlates with increased CD8 IFNγ+ tumor infiltrating T cells. These hu-CB-BRGS mice provide a relevant preclinical animal model to facilitate prioritization of hypothesis-driven combination immunotherapies. Electronic supplementary material The online version of this article (10.1186/s40425-019-0518-z) contains supplementary material, which is available to authorized users.

Biliary atresia (BA) is a neonatal T cell-mediated, inflammatory, sclerosing cholangiopathy. In the rhesus rotavirus (RRV)-induced neonatal mouse model of BA (murine BA), mice lacking B cells do not develop BA, and the lack of B cells is associated with loss of T-cell and macrophage activation. The aim of this study was to determine the mechanism of B cell-mediated immune activation (antigen presentation versus cytokine production) in murine BA. Normal neonatal B cells in the liver are predominantly at pro-B and pre-B cellular development. However, BA mice exhibit a significant increase in the number and activation status of mature liver B cells. Adoptively transferred B cells into RRV-infected, B cell-deficient mice were able to reinstate T-cell and macrophage infiltration and biliary injury. Nonetheless, neonatal liver B cells were incompetent at antigen presentation to T cells. Moreover, 3-83 immunoglobulin transgenic mice, in which B cells only present an irrelevant antigen, developed BA, indicating a B-cell antigen-independent mechanism. B cells from BA mice produced a variety of innate and adaptive immune cytokines associated with immune activation. In vitro trans-well studies revealed that BA B cells secreted cytokines that activated T cells based on increased expression of T-cell activation marker cluster of differentiation 69. Conclusion: Neonatal liver B cells are highly activated in murine BA and contribute to immune activation through production of numerous cytokines involved in innate and adaptive immunity; this work provides increased knowledge on the capacity of neonatal B cells to contribute to an inflammatory disease through cytokine-mediated mechanisms, and future studies should focus on targeting B cells as a therapeutic intervention in human BA.

The development and function of B lymphocytes is regulated by numerous signaling pathways, some emanating from the B-cell antigen receptor (BCR). The spleen tyrosine kinase (Syk) plays a central role in the activation of the BCR, but less is known about its contribution to the survival and maintenance of mature B cells. We generated mice with an inducible and B-cell-specific deletion of the Syk gene and found that a considerable fraction of mature Syk-negative B cells can survive in the periphery for an extended time. Syk-negative B cells are defective in BCR, RP105 and CD38 signaling but still respond to an IL-4, anti-CD40, CpG or LPS stimulus. Our in vivo experiments show that Syk-deficient B cells require BAFF receptor and CD19/PI3K signaling for their long-term survival. These studies also shed a new light on the signals regulating the maintenance of the normal mature murine B-cell pool.

Hematopoietic humanized mice (hu-mice) have been developed to study the human immune system in an experimental in vivo model, and experiments to improve its performance are ongoing. Previous studies have suggested that the impaired maturation of human B cells observed in hu-mice might be in part due to inefficient interaction of the human B-cell-activating factor (hBAFF) receptor with mouse B-cell-activating factor (mBAFF), as this cytokine is an important homeostatic and differentiation factor for B lymphocytes both in mice and humans. To investigate this hypothesis, we created a genetically engineered mouse strain in which a complementary DNA (cDNA) encoding full-length hBAFF replaces the mBAFF-encoding gene. Expression of hBAFF in the endogenous mouse locus did not lead to higher numbers of mature and effector human B cells in hu-mice. Instead, B cells from hBAFF knock-in (hBAFFKI) hu-mice were in proportion more immature than those of hu-mice expressing mBAFF. Memory B cells, plasmablasts, and plasma cells were also significantly reduced, a phenotype that associated with diminished levels of immunoglobulin G and T-cell-independent antibody responses. Although the reasons for these findings are still unclear, our data suggest that the inefficient B-cell maturation in hu-mice is not due to suboptimal bioactivity of mBAFF on human B cells.

Lysophospholipids have emerged as biologically important chemoattractants capable of directing lymphocyte development, trafficking, and localization. Lysophosphatidic acid (LPA) is a major lysophospholipid found systemically, and its levels are elevated in certain pathological settings, such as cancer and infections. In this study, we demonstrate that BCR signal transduction by mature murine B cells is inhibited upon LPA engagement of the LPA5 (GPR92) receptor via a Gα12/13-Arhgef1 pathway. The inhibition of BCR signaling by LPA5 manifests by impaired intracellular calcium store release and most likely by interfering with inositol 1,4,5-triphosphate receptor activity. We further show that LPA5 also limits Ag-specific induction of CD69 and CD86 expression and that LPA5-deficient B cells display enhanced Ab responses. Thus, these data show that LPA5 negatively regulates BCR signaling, B cell activation, and immune response. Our findings extend the influence of lysophospholipids on immune function and suggest that alterations in LPA levels likely influence adaptive humoral immunity.

Receptor editing, a major mechanism of B cell tolerance, can also lead to allelic inclusion at the immunoglobulin light chain loci and the development of B cells that co-express two different immunoglobulin light chains and, therefore, two antibody specificities. Most allelically included B cells express two κ chains, although rare dual-λ cells are also observed. Moreover, these cells typically co-express an autoreactive and a nonautoreactive antibody. Thus, allelically included B cells could operate like “Trojan horses”: expression and function of the nonautoreactive antigen receptors might promote their maturation, activation, and terminal differentiation into effector cells that also express and secrete autoantibodies. Indeed, dual-κ B cells are greatly expanded into effector B cell subsets in some autoimmune mice, thus indicating they might play an important role in disease.

Chronic infection with human immunodeficiency virus impairs B cell antigen receptor signaling via a newly described mechanism of inhibitory signaling mediated by IgG3 and the IgG Fc receptor FcRγIIb (CD32b).