CD8 T lymphocytes are able to eliminate nascent tumor cells through a process referred to as immune surveillance. 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 increased 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 6 distinct G-protein-coupled receptors and several of which are expressed by T cells, although the precise role of LPA signaling in CD8 T cell activation and function has not been defined. Here, we demonstrate 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.
Lysophosphatidic acid; LPA5; GPR92; CD8 T cells; melanoma
B lymphocytes are often considered a homogenous population. However, B cells in both mouse and humans are comprised of distinct subpopulations that differ in development, phenotype, function, and microenvironmental niches. Much of our understanding about how these different B-cells populations mount antibody responses has been derived from experimental findings in mouse models and based on the use of model antigens. These reductionist studies performed over decades have been invaluable in defining the parameters of the B-cell antibody response to different types of antigens. However, these antigens also are now known to differ in a significant manner from bona fide physiological pathogens, and precisely how these different B-cell subsets divide labor in the primary humoral immune defense of pathogens is less well understood. While there are no absolutes in this area, there are recurring themes that divide the roles of B-cell subsets to different arms of the antibody response. This review provides an overview of rules that govern the B-cell labor roles, exceptions that break these rules, and models that have been used to define them.
B-cell subpopulation; Follicular B cells; Marginal zone B cells; Primary antibody response
The development of an adaptive immune system based on the random generation of antigen receptors requires a stringent selection process that sifts through receptor specificities to remove those reacting with self-antigens. In the B-cell lineage, this selection process is first applied to IgM+ immature B cells. By using increasingly sophisticated mouse models, investigators have identified the central tolerance mechanisms that negatively select autoreactive immature B cells and prevent inclusion of their antigen receptors into the peripheral B-cell pool. Additional studies have uncovered mechanisms that promote the differentiation of nonautoreactive immature B cells and their positive selection into the peripheral B-cell population. These mechanisms of central selection are fundamental to the generation of a naïve B-cell repertoire that is largely devoid of self-reactivity while capable of reacting with any foreign insult.
Nave B cells must react with foreign insult but lack self-reactivity. Experiments in mice have defined selection processes that determine the entry of new B cells into the peripheral B-cell population.
The hematopoietic humanized mouse (hu-mouse) model is a powerful resource to study and manipulate the human immune system. However, a major and recurrent issue with this model has been the poor maturation of B cells that fail to progress beyond the transitional B cell stage. Interestingly, a similar problem has been reported in transplant patients that receive cord blood stem cells. In this study, we characterize the development of human B and T cells in the lymph nodes (LNs) and spleen of BALB/c-Rag2nullIl2rγnull hu-mice. We find a dominant population of immature B cells in the blood and spleen early, followed by a population of human T cells, coincident with the detection of LNs. Notably, in older mice we observe a major population of mature B cells in LNs and in the spleens of mice with higher T cell frequencies. Moreover, we demonstrate that the T cells are necessary for B cell maturation, as introduction of autologous human T cells expedites the appearance of mature B cells, while in vivo depletion of T cells retards B cell maturation. The presence of the mature B cell population correlates with enhanced IgG and Ag-specific responses to both T-dependent and T-independent challenges, indicating their functionality. These findings enhance our understanding of human B cell development, provide increased details of the reconstitution dynamics of hu-mice, and validates the use of this animal model to study mechanisms and treatments for the similar delay of functional B cells associated with cord blood transplantations.
Immature B cells are generated daily in the bone marrow tissue. More than half of the newly generated immature B cells are autoreactive and bind a self-antigen, while the others are nonautoreactive. A selection process has evolved on the one hand to thwart development of autoreactive immature B cells and, on the other hand, to promote further differentiation of nonautoreactive immature B cells into transitional and mature B cells. These negative and positive selection events are carefully regulated by signals that emanate from the antigen receptor, whether antigen-mediated or tonic, and are influenced by signals that are generated by receptors that bind cytokines, chemokines, and other factors produced in the bone marrow tissue. These signals, therefore, are the predominant driving forces for the generation of a B cell population that is capable of protecting the body from infections while maintaining self-tolerance. Here, we review recent findings from our group and others that describe how tonic antigen receptor signaling and bone marrow cytokines regulate the selection of immature B cells.
B cell development; B cell selection; B cell tolerance; Bone marrow; BCR signaling; Cytokine
A leading theory regarding the pathogenesis of biliary atresia (BA) is that bile duct injury is initiated by a virus infection, followed by an autoimmune response targeting bile ducts. In experimental models of autoimmune diseases, B cells have been shown to play an important role. The aim of this study was to determine the role of B cells in the development of biliary obstruction in the Rhesus rotavirus (RRV)-induced mouse model of BA. Wild-type (WT) and B cell-deficient (Ig-α-/-) mice received RRV shortly after birth. Ig-α-/- RRV-infected mice had significantly increased disease-free survival rate compared to WT RRV-infected BA mice (76.8% vs. 17.5%). In stark contrast to the RRV-infected BA mice, the RRV-infected Ig-α-/- mice did not have hyperbilirubinemia or bile duct obstruction. The RRV-infected Ig-α-/- mice had significantly less liver inflammation and Th1 cytokine production compared to RRV-infected WT mice. In addition, Ig-α-/- mice had significantly increased numbers of regulatory T cells (Tregs) at baseline and after RRV infection compared to WT mice. However, depletion of Tregs in Ig-α-/- mice did not induce biliary obstruction, indicating that the expanded Tregs in the Ig-α-/- mice were not the sole reason for protection from disease. Conclusion: B cell deficient Ig-α-/- mice are protected from biliary obstruction in the RRV-induced mouse model of BA, indicating a primary role of B cells in mediating disease pathology. The mechanism of protection may involve lack of B cell antigen presentation, which impairs T-cell activation and Th1 inflammation. Immune modulators that inhibit B cell function may be a new strategy for treatment of BA.
Dual–light chain–expressing B cells in autoimmune prone mice increase with age, contribute to the memory and plasma cell compartments, and are autoreactive.
Rare dual-reactive B cells expressing two types of Ig light or heavy chains have been shown to participate in immune responses and differentiate into IgG+ cells in healthy mice. These cells are generated more often in autoreactive mice, leading us to hypothesize they might be relevant in autoimmunity. Using mice bearing Igk allotypic markers and a wild-type Ig repertoire, we demonstrate that the generation of dual-κ B cells increases with age and disease progression in autoimmune-prone MRL and MRL/lpr mice. These dual-reactive cells express markers of activation and are more frequently autoreactive than single-reactive B cells. Moreover, dual-κ B cells represent up to half of plasmablasts and memory B cells in autoimmune mice, whereas they remain infrequent in healthy mice. Differentiation of dual-κ B cells into plasmablasts is driven by MRL genes, whereas the maintenance of IgG+ cells is partly dependent on Fas inactivation. Furthermore, dual-κ B cells that differentiate into plasmablasts retain the capacity to secrete autoantibodies. Overall, our study indicates that dual-reactive B cells significantly contribute to the plasmablast and memory B cell populations of autoimmune-prone mice suggesting a role in autoimmunity.
Antibody diversity is generated by a random gene recombination process with the inherent risk of the production of autoreactive specificities. The current view suggests that B cells expressing such specificities are negatively selected at an early developmental stage. Using the knock-in model system of the 3–83 autoreactive B-cell antigen receptor (BCR) in combination with precursor-BCR (pre-BCR) deficiency, we show here that the 3–83 BCR mediates efficient generation of B cells in the presence, but not the absence, of a strongly recognized auto-antigen. Experiments with mixed bone marrow chimeras showed that combining the 3–83 BCR with the corresponding auto-antigen resulted in efficient reconstitution of B-cell development in immune-deficient mice. These results suggest that B cells are positively selected by recognition of self-antigens during developmental stages that precede receptor editing. Moreover, the data indicate that the pre- BCR functions as a specialized autoreactive BCR to initiate positive selection at a stage where the cells express immunoglobulin heavy but not light chains.
Autoimmune; Proliferation; Self-reactive; Selection
Exposure to second hand tobacco smoke is associated with the development and/or exacerbation of several different pulmonary diseases in humans. To better understand the possible effects of second hand smoke exposure in humans, we sub-chronically (4 weeks) exposed mice to a mixture of mainstream and sidestream tobacco smoke at concentrations similar to second hand smoke exposure in humans. The inflammatory response to smoke exposures was assessed at the end of this time by enumeration of pulmonary leukocyte infiltration together with measurements of lung elastance and pathology. This response was measured in both healthy wild type (C57BL/6) mice as well as mouse mutants deficient in the expression of Arhgef1 (Arhgef1−/−) that display constitutive pulmonary inflammation and decreased lung elastance reminiscent of emphysema. The results from this study show that sub-chronic second hand smoke exposure leads to significantly increased numbers of airspace leukocytes in both healthy and mutant animals. While sub-chronic cigarette smoke exposure is not sufficient to induce changes in lung architecture as measured by mean linear intercept, both groups exhibit a significant decrease in lung elastance. Together these data demonstrate that even sub-chronic exposure to second hand smoke is sufficient to induce pulmonary inflammation and decrease lung elastance in both healthy and diseased animals and in the absence of tissue destruction.
second hand smoke; inflammation; lung mechanics
Hematopoietic humanized mice generated via transplantation of human hematopoietic stem cells (hHSCs) into immunodeficient mice are a valuable tool for studying development and function of the human immune system. This study was performed to generate a protocol that improves development and quality of humanized mice in the BALB/c-Rag2nullIl2rγnull strain, testing route of injection, in vitro culture and freezing of hHSCs, types of cytokines in the culture, and co-injection of lineage-depleted CD34− cells. Specific hHSC culturing conditions and the addition of support cells were found to increase the frequency, and human hematopoietic chimerism, of humanized mice. The optimized protocol resulted in BALB/c-Rag2nullIl2rγnull humanized mice displaying more consistent human hematopoietic and lymphoid engraftment. Thus, hematopoietic humanized mice generated on a BALB/c immunodeficient background represent a useful model to study the human immune system.
hematopoietic stem cell; human cord blood; hematopoietic chimerism; immunodeficient mouse; cytokine; humanized mouse
The Glucocorticoid-Induced Tumor necrosis factor Receptor GITR, a member of the tumor necrosis factor receptor superfamily, has been shown to be important in modulating immune responses in the context of T cell immunity. B lymphocytes also express GITR, but a role of GITR in humoral immunity has not been fully explored. To address this question, we performed studies to determine the kinetics of GITR expression on naïve and stimulated B cells and the capacity of B cells to develop and mount antibody responses in GITR−/− mice. Results of our studies indicate that all mature B cells express GITR on the cell surface, albeit at different levels. Expression of GITR on naïve mature B cells is upregulated by BCR signaling, but is counteracted by helper T cell-related factors and other inflammatory signals in vitro. In line with these findings, expression of GITR on germinal center and memory B cells is lower than that on naïve B cells. However, the expression of GITR is strongly upregulated in plasma cells. Despite these differences in GITR expression, the absence of GITR has no effect on T cell-dependent and T cell-independent antibody responses to model antigens in GITR−/− mice, or on B cell activation and proliferation in vitro. GITR deficiency manifests only with a slight reduction of mature B cell numbers and increased turnover of naïve B cells, suggesting that GITR slightly contributes to mature B cell homeostasis. Overall, our data indicate that GITR does not play a significant role in B cell development and antibody responses to T-dependent and independent model antigens within the context of a GITR-deficient genetic background.
BAFF is an important pro-survival cytokine for mature B cells. However, previous studies have shown that the BAFF receptor, BAFF-R, is already expressed at the immature B cell stage, and that the pro-survival protein Bcl-2 does not completely complement the B cell defects resulting from the absence of BAFF-R or BAFF. Thus, we hypothesized that BAFF also functions to aid the differentiation of non-autoreactive immature B cells into transitional B cells and to promote their positive selection. We found that BAFF-R is expressed at higher levels on non-autoreactive than autoreactive immature B cells and that its expression correlates with that of surface IgM and with tonic BCR signaling. Our data indicate that BAFF-R signaling enhances the generation of transitional CD23− B cells in vitro by increasing cell survival. In vivo, however, BAFF-R signaling is dispensable for the generation of CD23− transitional B cells in the bone marrow, but is important for the development of transitional CD23− T1 B cells in the spleen. In addition, we show that BAFF is essential for the differentiation of CD23− into CD23+ transitional B cells both in vitro and in vivo through a mechanism distinct from that mediating cell survival, but requiring tonic BCR signaling. In summary, our data indicate that BAFF-R and tonic BCR signals cooperate to enable non-autoreactive immature B cells to differentiate into transitional B cells and to be positively selected into the naïve B cell repertoire.
Rfv3 is an autosomal dominant gene that influences the recovery of resistant mice from Friend retrovirus (FV) infection by limiting viremia and promoting a more potent neutralizing antibody response. We previously reported that Rfv3 is encoded by Apobec3, an innate retrovirus restriction factor. However, it was recently suggested that the Rfv3 susceptible phenotype of high viremia at 28 days postinfection (dpi) was more dominantly controlled by the B-cell-activating factor receptor (BAFF-R), a gene that is linked to but located outside the genetically mapped region containing Rfv3. Although one prototypical Rfv3 susceptible mouse strain, A/WySn, indeed contains a dysfunctional BAFF-R, two other Rfv3 susceptible strains, BALB/c and A.BY, express functional BAFF-R genes, determined on the basis of genotyping and B-cell immunophenotyping. Furthermore, transcomplementation studies in (C57BL/6 [B6] × BALB/c)F1 and (B6 × A.BY)F1 mice revealed that the B6 Apobec3 gene significantly influences recovery from FV viremia, cellular infection, and disease at 28 dpi. Finally, the Rfv3 phenotypes of prototypic B6, A.BY, A/WySn, and BALB/c mouse strains correlate with reported Apobec3 mRNA expression levels. Overall, these findings argue against the generality of BAFF-R polymorphisms as a dominant mechanism to explain the Rfv3 recovery phenotype and further strengthen the evidence that Apobec3 encodes Rfv3.
Humoral immunity to viruses and encapsulated bacteria is comprised of T cell–independent type 2 (TI-2) antibody responses that are characterized by rapid antibody production by marginal zone and B1 B cells. We demonstrate that toll-like receptor (TLR) ligands influence the TI-2 antibody response not only by enhancing the overall magnitude but also by skewing this response to one that is dominated by IgG isotypes. Importantly, TLR ligands facilitate this response by inducing type I interferon (IFN), which in turn elicits rapid and significant amounts of antigen-specific IgG2c predominantly from FO (follicular) B cells. Furthermore, we show that although the IgG2c antibody response requires B cell–autonomous IFN-α receptor signaling, it is independent of B cell–intrinsic TLR signaling. Thus, innate signals have the capacity to enhance TI-2 antibody responses by promoting participation of FO B cells, which then elaborate effective IgG anti-pathogen antibodies.
B cell receptors (BCRs) generate tonic signals critical for B cell survival and early B cell development. To determine whether these signals also mediate the development of transitional and mature B cells, we examined B cell development using a mouse strain in which nonautoreactive immunoglobulin heavy and light chain–targeted B cells express low surface BCR levels. We found that reduced BCR expression translated into diminished tonic BCR signals that strongly impaired the development of transitional and mature B cells. Constitutive expression of Bcl-2 did not rescue the differentiation of BCR-low B cells, suggesting that this defect was not related to decreased cell survival. In contrast, activation of the Ras pathway rescued the differentiation of BCR-low immature B cells both in vitro and in vivo, whereas extracellular signal-regulated kinase (Erk) inhibition impaired the differentiation of normal immature B cells. These results strongly suggest that tonic BCR signaling mediates the differentiation of immature into transitional and mature B cells via activation of Erk, likely through a pathway requiring Ras.
During B cell development, immature B cell fate is determined by whether the B cell antigen receptor is engaged in the bone marrow. Immature B cells that are non-autoreactive continue maturation and emigrate from the marrow whereas autoreactive immature B cells remain and are tolerized. However, the microenvironment where these events occur and the chemoattractants responsible for immature B cell trafficking within and out of the bone marrow remain largely undefined. Sphingosine 1-phosphate (S1P) is a chemoattractant that directs lymphocyte trafficking and thymocyte egress and in this study we investigated whether S1P contributed to B cell development, egress and positioning within the bone marrow. Our findings show that immature B cells are chemotactic towards S1P but that this response is dependent on antigen receptor specificity: non-autoreactive, but not autoreactive, immature B cells migrate towards S1P and are shown to require S1P3 receptor for this response. Despite this response, S1P3 is shown not to facilitate immature B cell egress but is required for normal B cell development including the positioning of transitional B cells within bone marrow sinusoids. These data indicate that S1P3 signaling directs immature B cells to a bone marrow microenvironment important for both tolerance induction and maturation.
Immature B cells; Autoreactive; Sphingosine 1-phosphate; Migration
Lsc is a hematopoietic-restricted protein that functions as an effector of Gα12/13-associated G-protein coupled receptors that activates RhoA. In the absence of Lsc leukocytes exhibit impaired migration and B lymphocytes inefficiently resolve integrin-mediated adhesion. Here, we demonstrate that Lsc exists physiologically in primary B lymphocytes as a large molecular weight complex resembling a homo-tetramer. Interfering with the assembly of this large molecular weight Lsc oligomer results in the activation of both Lsc functional activities and leads to cell rounding and inhibition of integrin-mediated adhesion. During cell migration on integrin ligands we find Lsc localizes predominantly toward the rear of migrating cells where we suggest it activates RhoA to resolve integin-mediated adhesion. Together these data demonstrate that Lsc regulates integrin-mediated adhesive events at the trailing edge of migrating cells.
Lsc; oligomerization; RhoA; integrin; adhesion
Rationale: Arhgef1 is an intracellular protein, expressed by hematopoietic cells, that regulates signaling by both G protein–coupled receptors and RhoA, and, consequently, is required for appropriate migration and adhesion of diverse leukocyte populations.
Objectives: To evaluate a possible contribution for Arhgef1 in the development of airway inflammation and airway hyperreactivity.
Methods: Arhgef1-deficient (Arhgef1−/−) and wild-type (WT) mice were sensitized and airway challenged, followed by measurement of airway responsiveness to inhaled methacholine. Inflammation was assessed by several parameters that included flow cytometric analysis and histology. Arhgef1-deficient recipients were reconstituted with WT T lymphocytes before sensitization and challenge, and again measured for airway responsiveness and inflammation. Cytokine production in response to specific antigen was measured in cultures of isolated leukocytes from lung and spleen and compared with the levels generated in lung and spleen explant cultures.
Measurements and Main Results: Arhgef1−/− mice display significantly reduced airway hyperreactivity, Th2 cytokine production, and lung inflammation, despite intact systemic immunity. After airway challenge of Arhgef1−/− mice, antigen-specific T cells were present in mutant lungs, but were found to interact with CD11c+ cells at a significantly reduced frequency. Adoptive transfer of WT T cells into Arhgef1−/− mice restored airway hyperreactivity and inflammation.
Conclusions: These data demonstrate that T cells depend on Arhgef1 to promote lung inflammation. Moreover, a deficiency in Arhgef1 results in reduced T cell–CD11c+ antigen-presenting cell interaction, and likely underscores the inability of Arhgef1−/− mice to mount an adaptive immune response to airway challenge.
airway hyperreactivity; cytokines; lung inflammation; T cells
Early B cell development is characterized by stepwise, ordered rearrangement of the immunoglobulin (Ig) heavy (HC) and light (LC) chain genes. Only one of the two alleles of these genes is used to produce a receptor, a phenomenon referred to as allelic exclusion. It has been suggested that pre–B cell receptor (pre-BCR) signals are responsible for down-regulation of the VDJH-recombinase machinery (Rag1, Rag2, and terminal deoxynucleotidyl transferase [TdT]), thereby preventing further rearrangement on the second HC allele. Using a mouse model, we show that expression of an inducible μHC transgene in Rag2−/− pro–B cells induces down-regulation of the following: (a) TdT protein, (b) a transgenic green fluorescent protein reporter reflecting endogenous Rag2 expression, and (c) Rag1 primary transcripts. Similar effects were also observed in the absence of surrogate LC (SLC) components, but not in the absence of the signaling subunit Ig-α. Furthermore, in wild-type mice and in mice lacking either λ5, VpreB1/2, or the entire SLC, the TdT protein is down-regulated in μHC+LC− pre–B cells. Surprisingly, μHC without LC is expressed on the surface of pro–/pre–B cells from λ5−/−, VpreB1−/−VpreB2−/−, and SLC−/− mice. Thus, SLC or LC is not required for μHC cell surface expression and signaling in these cells. Therefore, these findings offer an explanation for the occurrence of HC allelic exclusion in mice lacking SLC components.
B cell development; allelic exclusion; Rag; TdT; pre-BCR
In mouse mutants incapable of expressing μ chains, VκJκ joints are detected in the CD43+ B cell progenitors. In agreement with these earlier results, we show by a molecular single cell analysis that 4–7% of CD43+ B cell progenitors in wild-type mice rearrange immunoglobulin (Ig)κ genes before the assembly of a productive VHDHJH joint. Thus, μ chain expression is not a prerequisite to Igκ light chain gene rearrangements in normal development. Overall, ∼15% of the total CD43+ B cell progenitor population carry Igκ gene rearrangements in wild-type mice. Together with the results obtained in the mouse mutants, these data fit a model in which CD43+ progenitors rearrange IgH and Igκ loci independently, with a seven times higher frequency in the former. In addition, we show that in B cell progenitors VκJκ joining rapidly initiates κ chain expression, irrespective of the presence of a μ chain.
B cell development; bone marrow; immunoglobulin gene rearrangement