Signaling lymphocytic activation molecule-associated protein controls NKT cell functions

X-linked lymphoproliferative disease (XLP) is a fatal immunological disorder that typically manifests following EBV infection. XLP patients exhibit a number of immune defects including abnormal T, B, and NK lymphocyte function. These defects have been attributed to mutations of Src homology 2 domain-containing gene 1A (SH2D1A), the gene encoding signaling lymphocytic activation molecule-associated protein (SAP), an intracellular adaptor molecule expressed in lymphocytes. We have observed that SAP knockout (SAPKO) mice and humans with XLP have a complete lack of CD1d-restricted NKT cells. As expected, SAPKO mice injected with the NKT cell agonist, alpha-galactosylceramide failed to generate NKT cell IFN-gamma or IL-4. Furthermore, in contrast to wild-type littermates, SAPKO mice coinjected with OVA and alpha-galactosylceramide failed to mount OVA-specific CTL responses. These data suggest that an absence of NKT cells may underlie part of the immune dysregulation seen in SAPKO mice and in XLP patients.     

Perturbation of B cell activation in SLAM-associated protein-deficient mice is associated with changes in gammaherpesvirus latency reservoirs

Signaling lymphocyte activation molecule (SLAM)-associated protein (SAP)) interactions with SLAM family proteins play important roles in immune function. SAP-deficient mice have defective B cell function, including impairment of germinal center formation, production of class-switched Ig, and development of memory B cells. B cells are the major reservoir of latency for both EBV and the homologous murine gammaherpesvirus, gammaherpesvirus 68. There is a strong association between the B cell life cycle and viral latency in that the virus preferentially establishes latency in activated germinal center B cells, which provides access to memory B cells, a major reservoir of long-term latency. In the current studies, we have analyzed the establishment and maintenance of gammaHV68 latency in wild-type and SAP-deficient mice. The results show that, despite SAP-associated defects in germinal center and memory B cell formation, latency was established and maintained in memory B cells at comparable frequencies to wild-type mice, although the paucity of memory B cells translated into a 10-fold reduction in latent load. Furthermore, there were defects in normal latency reservoirs within the germinal center cells and IgD(+)"naive" B cells in SAP-deficient mice, showing a profound effect of the SAP mutation on latency reservoirs.     

Defective B1 cell homing to the peritoneal cavity and preferential recruitment of B1 cells in the target organs in a murine model for systemic lupus erythematosus

We previously reported that B lymphocyte chemoattractant (BLC; CXCL13) was highly and ectopically expressed in aged (NZB x NZW)F1 (BWF1) mice developing lupus nephritis, and that B1 cells were preferentially chemoattracted toward BLC. We demonstrate in this study that B1 cells fail to home to the peritoneal cavity in aged BWF1 mice developing lupus nephritis, and that they are preferentially recruited to the target organs including the kidney, lung, and thymus when injected i.v. In contrast, B1 cells homed to the peritoneal cavity in aged BALB/c mice as effectively as in young mice. Accumulation of B1 cells to the omentum milky spots was also impaired in aged BWF1 mice compared with young mice. CD11bhighF4/80high cells with macrophage morphology were confirmed to be a major cell source for BLC in the peritoneal cavity both in young and aged BWF1 mice. However, the number of BLC-producing peritoneal macrophages was markedly decreased in aged BWF1 mice. These results suggest that the decreased number of BLC-producing peritoneal macrophages together with ectopic high expression of BLC in aged BWF1 mice result in abnormal B1 cell trafficking during the development of murine lupus.     

Lack of B cell leakiness in BALB/cA-nu, scid double mutant mice.

BALB/cA mice homozygous for both nu and scid mutations (BALB/cA-nu/nu, scid/scid) were developed by mating between BALB/cA-scid and BALB/cA-nu. These mice have greater longevity than C.B-17-scid because no thymic lymphoma occurs in them unlike in the latter. C.B-17-scid is known to show the leaky phenomenon in which a few clones of functional T and B cells develop in aged C.B-17-scid. Unexpectedly, the leaky B cells and T cells were absent or suppressed in BALB/cA-nu, scid mice when cytokine expressions were determined by RT-PCR, lymphocyte phenotypes by flow cytometry and serum immunoglobulin levels by ELISA. These results indicate that B cell leakiness may be induced by leaked T cells. BALB/cA-nu, scid mice may be useful as a recipient in allo- and xeno-transplantation experiments because of the absence of both thymic lymphomas and leakiness, in addition to lack of hair.     

Age-Related Decrease of Meiotic Cohesins in Human Oocytes

Aneuploidy in fetal chromosomes is one of the causes of pregnancy loss and of congenital birth defects. It is known that the frequency of oocyte aneuploidy increases with the human maternal age. Recent data have highlighted the contribution of cohesin complexes in the correct segregation of meiotic chromosomes. In mammalian oocytes, cohesion is established during the fetal stages and meiosis-specific cohesin subunits are not replenished after birth, raising the possibility that the long meiotic arrest of oocytes facilitates a deterioration of cohesion that leads to age-related increases in aneuploidy. We here examined the cohesin levels in dictyate oocytes from different age groups of humans and mice by immunofluorescence analyses of ovarian sections. The meiosis-specific cohesin subunits, REC8 and SMC1B, were found to be decreased in women aged 40 and over compared with those aged around 20 years (P<0.01). Age-related decreases in meiotic cohesins were also evident in mice. Interestingly, SMC1A, the mitotic counterpart of SMC1B, was substantially detectable in human oocytes, but little expressed in mice. Further, the amount of mitotic cohesins of mice slightly increased with age. These results suggest that, mitotic and meiotic cohesins may operate in a coordinated way to maintain cohesions over a sustained period in humans and that age-related decreases in meiotic cohesin subunits impair sister chromatid cohesion leading to increased segregation errors.     

Expression of LIF in transgenic mice results in altered thymic epithelium and apparent interconversion of thymic and lymph node morphologies.

Leukemia inhibitory factor (LIF) is a cytokine involved in embryonic and hematopoietic development. To investigate the effects of LIF on the lymphoid system, we generated a line of transgenic mice that expresses diffusible LIF protein specifically in T cells. These mice display two categories of phenotype that were not previously attributed to LIF overexpression. First, they display B cell hyperplasia, polyclonal hypergammaglobulinemia and mesangial proliferative glomerulonephritis, defects similar to those described for transgenic mice overexpressing the functionally related cytokine, interleukin-6. Secondly, the LIF transgenic mice display novel thymic and lymph node abnormalities. In the thymus, cortical CD4+CD8+ lymphocytes are lost, while numerous B cell follicles develop. Peripheral lymph nodes contain a vastly expanded CD4+CD8+ lymphocyte population. Furthermore, the thymic epithelium is profoundly disorganized, suggesting that disruption of stroma-lymphocyte interactions is responsible for many observed defects. Transplantation of transgenic bone marrow into wild type recipients transfers both the thymic and lymph node defects. However, transplantation of wild type marrow into transgenic recipients rescues the lymph node abnormality, but not the thymic defect, indicating the thymic epithelium is irreversibly altered. Our observations are consistent with a role for LIF in maintaining a functional thymic epithelium that will support proper T cell maturation.     

I-kappa B kinase beta is critical for B cell proliferation and antibody response.

The NF-kappaB proteins are critical in the regulation of the immune and inflammatory response. Stimulation of the NF-kappaB pathway leads to increases in I-kappaB kinase beta (IKKbeta) kinase activity to result in the enhanced phosphorylation and degradation of I-kappaB and the translocation of the NF-kappaB proteins from the cytoplasm to the nucleus. In this study, a dominant-negative IKKbeta mutant expressed from the IgH promoter was used to generate transgenic mice to address the role of IKKbeta on B cell function. Although these transgenic mice were defective in activating the NF-kappaB pathway in B cells, they exhibited no defects in B lymphocyte development or basal Ig levels. However, they exhibited defects in the cell cycle progression and proliferation of B cells in response to treatment with LPS, anti-CD40, and anti-IgM. Furthermore, selective defects in the production of specific Ig subclasses in response to both T-dependent and T-independent Ags were noted. These results suggest that IKKbeta is critical for the proliferation of B cells and the control of some aspects of the humoral response.     

Identifying the Role of E2 Domains on Alphavirus Neutralization and Protective Immune Responses

Background

Chikungunya virus (CHIKV) and other alphaviruses are the etiologic agents of numerous diseases in both humans and animals. Despite this, the viral mediators of protective immunity against alphaviruses are poorly understood, highlighted by the lack of a licensed human vaccine for any member of this virus genus. The alphavirus E2, the receptor-binding envelope protein, is considered to be the predominant target of the protective host immune response. Although envelope protein domains have been studied for vaccine and neutralization in flaviviruses, their role in alphaviruses is less characterized. Here, we describe the role of the alphavirus E2 domains in neutralization and protection through the use of chimeric viruses.

Methodology/Principal Findings

Four chimeric viruses were constructed in which individual E2 domains of CHIKV were replaced with the corresponding domain from Semliki Forest virus (SFV) (ΔDomA/ΔDomB/ΔDomC/ ΔDomA+B). Vaccination studies in mice (both live and inactivated virus) revealed that domain B was the primary determinant of neutralization. Neutralization studies with CHIKV immune serum from humans were consistent with mouse studies, as ΔDomB was poorly neutralized.

Conclusions/Significance

Using chimeric viruses, it was determined that the alphavirus E2 domain B was the critical target of neutralizing antibodies in both mice and humans. Therefore, chimeric viruses may have more relevance for vaccine discovery than peptide-based approaches, which only detect linear epitopes. This study provides new insight into the role of alphavirus E2 domains on neutralization determinants and may be useful for the design of novel therapeutic technologies.     

Expression of cross-reactive, shared idiotypes on anti-SEA antibodies from humans and mice with schistosomiasis

Immunoaffinity-purified antibodies against Schistosoma mansoni soluble egg Ag (SEA) from infected patients' sera differ idiotypically according to the donor's clinical form of the disease. The Id differ both by their ability to stimulate proliferation of anti-Id T cells and their recognition by anti-Id-specific sera. Also, mice infected with S. mansoni develop anti-Id T and B cell responses against mouse anti-SEA antibodies. We now show that anti-SEA antibodies from serum pools of chronic, but asymptomatic patients (AM1 and AM5) stimulate proliferation of spleen cells from mice infected with S. mansoni. However, AM8, anti-SEA antibodies from hepatosplenic patients, did not stimulate these spleen cells. The murine responses directly parallel patient studies where AM1 and AM5 Id-stimulated human PBMC, but AM8 Id did not. In competitive ELISA, using AM1 or AM-5-specific rabbit antisera or human anti-SEA mAb E5-specific rabbit antiserum, sera from mice infected for 8 and 16 wk (but not from uninfected mice) compete with AM1, AM5, or E5. These sera do not compete in the AM8/anti-AM8 competitive ELISA. Sera from 8-wk-infected mice inhibit more against AM1, AM5, and E5 than do sera from later infections, and anti-SEA immunoaffinity-purified antibodies from 8-wk-infected mice stimulate spleen cells from infected mice more than anti-SEA antibodies from sera of mice late in infection. However, spleen cells from more chronically infected mice are more responsive to either the murine or human anti-SEA antibody preparations than cells from mice with earlier infections. Both the ELISA data and lymphocyte responses indicate that anti-SEA antibodies from mice infected with S. mansoni for 8 wk bear Id cross-reactive with those expressed on anti-SEA antibodies from humans with chronic, asymptomatic schistosomiasis, but not those from hepatosplenic patients.     

Absence of an essential role for thymic stromal lymphopoietin receptor in murine B-cell development

The murine cytokine thymic stromal lymphopoietin (TSLP) supports the development of B220+ IgM+ immature B cells and induces thymocyte proliferation in vitro. Human TSLP, by contrast, activates CD11c+ dendritic cells, but not B or T cells. Recent studies have demonstrated that the receptor for TSLP consists of a heterodimer of the interleukin 7 (IL-7) alpha chain and a novel protein that resembles the hematopoietic cytokine receptor common gamma chain. We examined signal transduction by the gamma-like chains using chimeric receptor proteins. The cytoplasmic domain of the human, but not of the murine, gamma-like chain, activates Jak2 and Stat5 and supports the proliferation of hematopoietic cell lines. In order to assess the role of the murine gamma-like chain in vivo, we generated gamma-like chain-deficient mice. Receptor-deficient mice are unresponsive to TSLP but exhibit no obvious phenotypic defects. In particular, hematopoietic cell development appeared normal. B-cell development, including the IgM+ compartment, was unaffected by loss of the TSLP pathway, as were T lymphopoiesis and lymphocyte proliferation in vitro. Cytokine receptors that utilize the common gamma chain signal through the lymphocyte-specific kinase Jak3. Mice deficient in Jak3 exhibit a SCID phenotype but harbor a residual B220+ splenic lymphocyte population. We demonstrate here that this residual lymphocyte population is lost in mice lacking both the gamma-like chain and Jak3.