B cell intrinsic TLR signals amplify but are not required for humoral immunity

Although innate signals driven by Toll-like receptors (TLRs) play a crucial role in T-dependent immune responses and serological memory, the precise cellular and time-dependent requirements for such signals remain poorly defined. To directly address the role for B cell–intrinsic TLR signals in these events, we compared the TLR response profile of germinal center (GC) versus naive mature B cell subsets. TLR responsiveness was markedly up-regulated during the GC reaction, and this change correlated with altered expression of the key adaptors MyD88, Mal, and IRAK-M. To assess the role for B cell–intrinsic signals in vivo, we transferred MyD88 wild-type or knockout B cells into B cell–deficient μMT mice and immunized recipient animals with 4-hydroxy-3-nitrophenylacetyl (NP) chicken gamma globulin. All recipients exhibited similar increases in NP-specific antibody titers during primary, secondary, and long-term memory responses. The addition of lipopolysaccharide to the immunogen enhanced B cell-intrinsic, MyD88-dependent NP-specific immunoglobulin (Ig)M production, whereas NP-specific IgG increased independently of TLR signaling in B cells. Our data demonstrate that B cell–intrinsic TLR responses are up-regulated during the GC reaction, and that this change significantly promotes antigen-specific IgM production in association with TLR ligands. However, B cell–intrinsic TLR signals are not required for antibody production or maintenance.     

FLICE-inhibitory protein is a key regulator of germinal center B cell apoptosis

Affinity maturation of the B cell response to antigen (Ag) takes place in the germinal centers (GCs) of secondary follicles. Two sequential molecular mechanisms underpin this process. First, the B cell repertoire is diversified through hypermutation of the immunoglobulin (Ig) variable region genes. Second, mutant B cell clones with improved affinity for Ag are positively selected by Ag and CD40 ligand (L). This selection step is contingent upon "priming" of GC B cells for apoptosis. The molecular means by which B cell apoptosis is initiated and controlled in the GC remains unclear. Here, we show that GC B cell apoptosis is preceded by the rapid activation of caspase-8 at the level of CD95 death-inducing signaling complex (DISC). We found that GC B cells ex vivo display a preformed inactive DISC containing Fas-associated death domain-containing protein (FADD), procaspase-8, and the long isoform of cellular FADD-like IL-1beta-converting enzyme-inhibitory protein (c-FLIP(L)) but not the CD95L. In culture, c-FLIP(L) is rapidly lost from the CD95 DISC unless GC B cells are exposed to the survival signal provided by CD40L. Our results suggest that (a) the death receptor signaling pathway is involved in the affinity maturation of antibodies, and (b) c-FLIP(L) plays an active role in positive selection of B cells in the GC.     

Recovery of humoral immunity is critical for successful antiviral therapy in disseminated mouse adenovirus type 1 infection

Severe adenovirus infections in transplant recipients undergoing immunosuppressive therapy are of increasing concern. Controversy exists on the contribution of antiviral therapy and the host immune response to recovery from these infections. Here, we established a systemic mouse adenovirus type 1 (MAV-1) infection in cyclophosphamide (CyP)-treated BALB/c mice. CyP was administered at 100 mg per kg of body weight every other day for 2, 3, or 4 weeks, thereby inducing general but reversible leukopenia, with a major suppression of the B-cell numbers and functionality that was more pronounced than that seen with T cells. The outcome of MAV-1 infection was dependent on the duration of CyP therapy, as the mice with the most severe immunosuppression were the most vulnerable to MAV-1-induced hemorrhagic enteritis and mortality. The protective effect of concomitant antiviral therapy with cidofovir depended on the level of immunosuppression. The combination of cidofovir treatment with the withdrawal of immunosuppression was the most successful regimen for increasing survival rates. Survival was clearly correlated with the clearance of virus and increased titers of MAV-1-specific antibodies in sera. In addition, the passive transfer of MAV-1-specific immunoglobulin G into MAV-1-infected SCID BALB/c mice caused a marked delay in mortality, the extent of the delay being dependent on the titer of MAV-1-specific antibodies. Based on the critical role of the humoral immune response in the early defense against disseminated adenovirus infection, the concomitant use of adenovirus-specific immunoglobulins and antiviral therapy should be considered for transplant patients at risk for severe adenovirus infections.     

TYK2 is a key regulator of the surveillance of B lymphoid tumors

Aberrant activation of the JAK-STAT pathway has been implicated in tumor formation; for example, constitutive activation of JAK2 kinase or the enforced expression of STAT5 induces leukemia in mice. We show here that the Janus kinase TYK2 serves an opposite function. Mice deficient in TYK2 developed Abelson-induced B lymphoid leukemia/lymphoma as well as TEL-JAK2-induced T lymphoid leukemia with a higher incidence and shortened latency compared with WT controls. The cell-autonomous properties of Abelson murine leukemia virus-transformed (A-MuLV-transformed) TYK2(-/-) cells were unaltered, but the high susceptibility of TYK2(-/-) mice resulted from an impaired tumor surveillance, and accordingly, TYK2(-/-) A-MuLV-induced lymphomas were easily rejected after transplantation into WT hosts. The increased rate of leukemia/lymphoma formation was linked to a decreased in vitro cytotoxic capacity of TYK2(-/-) NK and NKT cells toward tumor-derived cells. RAG2/TYK2 double-knockout mice succumbed to A-MuLV-induced leukemia/lymphoma faster than RAG2(-/-)TYK2(+/-) mice. This defines NK cells as key players in tumor surveillance in Abelson-induced malignancies. Our observations provide compelling evidence that TYK2 is an important regulator of lymphoid tumor surveillance.     

Cdc42 and Rab8a are critical for intestinal stem cell division, survival, and differentiation in mice

The constant self renewal and differentiation of adult intestinal stem cells maintains a functional intestinal mucosa for a lifetime. However, the molecular mechanisms that regulate intestinal stem cell division and epithelial homeostasis are largely undefined. We report here that the small GTPases Cdc42 and Rab8a are critical regulators of these processes in mice. Conditional ablation of Cdc42 in the mouse intestinal epithelium resulted in the formation of large intracellular vacuolar structures containing microvilli (microvillus inclusion bodies) in epithelial enterocytes, a phenotype reminiscent of human microvillus inclusion disease (MVID), a devastating congenital intestinal disorder that results in severe nutrient deprivation. Further analysis revealed that Cdc42-deficient stem cells had cell division defects, reduced capacity for clonal expansion and differentiation into Paneth cells, and increased apoptosis. Cdc42 deficiency impaired Rab8a activation and its association with multiple effectors, and prevented trafficking of Rab8a vesicles to the midbody. This impeded cytokinesis, triggering crypt apoptosis and disrupting epithelial morphogenesis. Rab8a was also required for Cdc42-GTP activity in the intestinal epithelium, where continued cell division takes place. Furthermore, mice haploinsufficient for both Cdc42 and Rab8a in the intestine demonstrated abnormal crypt morphogenesis and epithelial transporter physiology, further supporting their functional interaction. These data suggest that defects of the stem cell niche can cause MVID. This hypothesis represents a conceptual departure from the conventional view of this disease, which has focused on the affected enterocytes, and suggests stem cell-based approaches could be beneficial to infants with this often lethal condition.     

TNF-alpha is critical for antitumor but not antiviral T cell immunity in mice

TNF-alpha antagonists are widely used in the treatment of inflammatory and autoimmune diseases, but their use is associated with reactivation of latent infections. This highlights the importance of TNF-alpha in immunity to certain pathogens and raises concerns that critical aspects of immune function are impaired in its absence. Unfortunately, the role of TNF-alpha in the regulation of T cell responses is clouded by a myriad of contradictory reports. Here, we show a role for TNF-alpha and its receptors, TNFR1 and TNFR2, specifically in antitumor immunity. TNF-alpha-deficient mice exhibited normal antiviral responses associated with strong inflammation. However, TNF-alpha/TNFR1-mediated signals on APCs and TNF-alpha/TNFR2 signals on T cells were critically required for effective priming, proliferation, and recruitment of tumor-specific T cells. Furthermore, in the absence of TNF-alpha signaling, tumor immune surveillance was severely abrogated. Finally, treatment with a CD40 agonist alone or in combination with TLR2 stimuli was able to rescue proliferation of TNF-alpha-deficient T cells. Therefore, TNF-alpha signaling may be required only for immune responses in conditions of limited immunostimulatory capacity, such as tumor surveillance. Importantly, these results suggest that prolonged continuous TNF-alpha blockade in patients may have long-term complications, including potential tumor development or progression.     

The role of CXCR4 in maintaining peripheral B cell compartments and humoral immunity

The chemokine receptor CXCR4 is expressed in B cells at multiple stages of their development. CXCR4 function in humoral immunity has not been fully investigated. We have generated gene-targeted mice in which CXCR4 can be selectively inactivated in B cells and have shown that it is required for retention of B cell precursors in the bone marrow. CXCR4-deficient B cell precursors that migrated prematurely became localized in splenic follicles despite their unresponsiveness to CXCL13. Concomitantly, mature B cell populations were reduced in the splenic marginal zone and primary follicles, and in the peritoneal cavity in the mutant animals, as were T-independent antibody responses. In addition, aberrant B cell follicles formed ectopically in intestinal lamina propria around Peyer's patches. These findings establish an important role for CXCR4 in regulating homeostasis of B cell compartmentalization and humoral immunity.     

Caspase activation is required for terminal erythroid differentiation

The cysteine proteases known as caspases play a central role in most apoptotic pathways. Here, we show that caspase inhibitors arrest the maturation of human erythroid progenitors at early stages of differentiation, before nucleus and chromatin condensation. Effector caspases such as caspase-3 are transiently activated through the mitochondrial pathway during erythroblast differentiation and cleave proteins involved in nucleus integrity (lamin B) and chromatin condensation (acinus)without inducing cell death and cleavage of GATA-1. These observations indicate a new function for caspases as key proteases in the process of erythroid differentiation.     

Genes required for B cell development

Mutations in a variety of genes can cause congenital agammaglobulinemia and a failure of B cell development. The currently known genes encode components of the pre-B cell receptor or proteins that are activated by cross-linking of the pre-B cell receptor. Defects in these genes result in a block in B cell differentiation at the pro-B to pre-B cell transition. A patient with a translocation involving a previously unknown gene, LRRC8, demonstrated a block at exactly the same point in B cell differentiation (see the related article beginning on page 1707). It will be interesting to determine whether the protein encoded by this gene interacts with the pre-B cell receptor signal transduction pathway or is involved in a new pathway.     

Cellular and humoral immunity in asymptomatic carriers of the hepatitis B virus

Eighty-eight asymptomatic HBsAg carriers were examined for cellular and humoral immunity and markers of virus B hepatitis in liver tissue and blood serum. The following results were obtained: HBsAg and HBcAg in liver tissue were found in 10 and 19 out of the 24 examinees, respectively, HBeAg and anti-Hbe in blood serum in 2 and 20 out of the 25 examinees, respectively. No alterations were recorded on the part of humoral immunity. Changes in cellular immunity manifested themselves by reduction in the number of T lymphocytes, and decreased response to PHA. Sensitization to HBsAg led to the purification of hepatitis B virus whereas the presence of simultaneous sensitization to liver-specific lipoprotein contributed to development of graver patterns of liver injury. The role of the immune system and hepatitis B virus itself in the pathogenesis of liver injury in asymptomatic HBsAg carriers is discussed.     

VSIG4, a B7 family-related protein, is a negative regulator of T cell activation

T cell activation by APCs is positively and negatively regulated by members of the B7 family. We have identified a previously unknown function for B7 family-related protein V-set and Ig domain-containing 4 (VSIG4). In vitro experiments using VSIG4-Ig fusion molecules showed that VSIG4 is a strong negative regulator of murine and human T cell proliferation and IL-2 production. Administration to mice of soluble VSIG4-Ig fusion molecules reduced the induction of T cell responses in vivo and inhibited the production of Th cell-dependent IgG responses. Unlike that of B7 family members, surface expression of VSIG4 was restricted to resting tissue macrophages and absent upon activation by LPS or in autoimmune inflammatory foci. The specific expression of VSIG4 on resting macrophages in tissue suggests that this inhibitory ligand may be important for the maintenance of T cell unresponsiveness in healthy tissues.     

Lymphocyte crosstalk is required for monocyte-intrinsic trained immunity to Plasmodium falciparum

Plasmodium falciparum (P. falciparum) induces trained innate immune responses in vitro, where initial stimulation of adherent PBMCs with P. falciparum–infected RBCs (iRBCs) results in hyperresponsiveness to subsequent ligation of TLR2. This response correlates with the presence of T and B lymphocytes in adherent PBMCs, suggesting that innate immune training is partially due to adaptive immunity. We found that T cell–depleted PBMCs and purified monocytes alone did not elicit hyperproduction of IL-6 and TNF-α under training conditions. Analysis of P. falciparum–trained PBMCs showed that DCs did not develop under control conditions, and IL-6 and TNF-α were primarily produced by monocytes and DCs. Transwell experiments isolating purified monocytes from either PBMCs or purified CD4⁺ T cells, but allowing diffusion of secreted proteins, enabled monocytes trained with iRBCs to hyperproduce IL-6 and TNF-α after TLR restimulation. Purified monocytes stimulated with IFN-γ hyperproduced IL-6 and TNF-α, whereas blockade of IFN-γ in P. falciparum–trained PBMCs inhibited trained responses. Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-Seq) on monocytes from patients with malaria showed persistently open chromatin at genes that appeared to be trained in vitro. Together, these findings indicate that the trained immune response of monocytes to P. falciparum is not completely cell intrinsic but depends on soluble signals from lymphocytes.     

B cell activation via CD40 is required for specific antibody production by antigen-stimulated human B cells

Costimulatory signals provided by T cells are required for B cells to produce specific antibody (Ab) to T-dependent antigen (Ag) bacteriophage phi x 174. In this study, we demonstrate that if cultured in the presence of anti-CD40, interleukin 10 (IL-10), and Ag, purified B cells can produce antiphage Ab in quantities comparable to those synthesized by B cells cocultured with Ag and T cells. Isotypes produced by B cells in this culture system correspond to those observed in sera of B cell donors. Culture of immunoglobulin (Ig)D- and IgD+ B cells reveals that Ag-induced production of antiphage Ab is restricted to IgD- subset of B cells. In the absence of Ag, anti-CD40/IL-10- stimulated B cells produce only minute amounts of antiphage Ab, indicating that Ag stimulation is indispensable and provides a signal that is synergistic with anti-CD40 and IL-10. Addition of a soluble form of the CD40 ligand (sgp39) to the culture system has a similar effect on specific Ab synthesis as anti-CD40; addition of the soluble construct, CD40 Ig, known to inhibit gp39/CD40 interaction, suppresses in vitro antiphage Ab production by Ag exposed peripheral blood mononuclear cells. Finally, in vivo requirement of gp39/CD40 interaction for specific Ab production was demonstrated by the finding that activated T cells from patients with x-linked hyper IgM syndrome express functionally defective gp39 and respond with depressed Ab titers and fail to switch from IgM to IgG after multiple phage immunizations. These observations illustrate that in vitro and possibly in vivo Ag-specific Ab synthesis requires the presence of Ag and IL-10, and activation signals via CD40.