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The forkhead DNA-binding protein FOXP3 is critical for the development and suppressive function of CD4(+)CD25(+) regulatory T cells (T(REG)), which play a key role in maintaining self-tolerance. Functionally, FOXP3 is capable of repressing transcription of cytokine genes regulated by NFAT. Various mechanisms have been proposed by which FOXP3 mediates these effects. Using novel cell lines that inducibly express either wild-type or mutant FOXP3, we have identified NFAT2 as an early target of FOXP3-mediated transcriptional repression. NFAT2 is typically expressed at low levels in resting T cells, but is up-regulated by NFAT1 upon cellular activation. We demonstrate that transcription from the NFAT2 promoter is significantly suppressed by FOXP3, and NFAT2 protein expression is markedly diminished in activated CD4(+)CD25(+)FOXP3(+) T(REG) compared with CD4(+)CD25(-)FOXP3(-) T cells. Chromatin immunoprecipitation experiments indicate that FOXP3 competes with NFAT1 for binding to the endogenous NFAT2 promoter. This antagonism of NFAT2 activity by FOXP3 is important for the anergic phenotype of T(REG), as ectopic expression of NFAT2 from a retroviral LTR partially restores expression of IL-2 in FOXP3(+) T(REG). These data suggest that FOXP3 functions not only to suppress the first wave of NFAT-mediated transcriptional responses, but may also affect sustained NFAT-mediated inflammatory gene expression through suppression of inducible NFAT2 transcription.

NKT cells comprise a separate T lineage expressing semi-invariant T cell receptors. Canonical invariant NKT (iNKT) cells specifically recognize lipid Ags presented by CD1d, a MHC class I-like molecule. iNKT cells function, in part, as initial responders to bacterial infection and play a role in immune surveillance and tumor rejection. The Wiskott-Aldrich Syndrome protein (WASp) serves as a crucial link between cellular stimuli and cytoskeletal rearrangements. Although we and others have identified a key role for WASp in homeostasis of T-regulatory and marginal zone B cells, little data exist regarding the role for WASp within the iNKT lineage. Analysis of WASp-expressing cell populations in heterozygous female WASp mice revealed a substantial selective advantage for WASp(+) vs WASp(-) iNKT cells. Although adult WASp-deficient (WASp(-/-)) mice had normal thymic and bone marrow iNKT numbers, we observed 2- to 3-fold reduction in the numbers of iNKT cells in the spleen and liver. This peripheral iNKT deficit is manifested, in part, due to defective iNKT homeostasis. WASp(-/-) iNKT cells exhibited reduced levels of integrin surface expression and decreased homing and/or retention within peripheral tissues in a competitive repopulation model. In addition, analysis of young mice showed that WASp is important for both maturation and egress of thymic iNKT cells. WASp(-/-) iNKT cells also exhibited a marked reduction in Ag-induced proliferation and cytokine production. Our findings highlight the crucial role for WASp in iNKT development, homeostasis, and activation, and identify iNKT dysfunction as an additional factor likely to contribute to the clinical features observed in WAS patients.

Foxp3 has been shown to be both necessary and sufficient for the development and function of naturally arising CD4+ CD25+ regulatory T cells in mice. Mutation of Foxp3 in Scurfy mice and FOXP3 in humans with IPEX results in fatal, early onset autoimmune disease and demonstrates the critical role of FOXP3 in maintaining immune homeostasis. The FOXP3 protein encodes several functional domains, including a C2H2 zinc finger, a leucine zipper, and a winged-helix/forkhead (FKH) domain. We have shown previously that FOXP3 functions as a transcriptional repressor and inhibits activation-induced IL-2 gene transcription. To characterize the role of each predicted functional domain on the in vivo activity of FOXP3, we have evaluated the location of point mutations identified in a large cohort of patients with the immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) and found them to cluster primarily within the FKH domain and the leucine zipper, but also present within the poorly defined N-terminal portion of the protein. The molecular functions of each of the IPEX-targeted domains were investigated. We show that FOXP3 is constitutively localized to the nucleus and this localization requires sequences at both the amino and C-terminal ends of its FKH domain. Moreover, FOXP3 was found to homodimerize through its leucine zipper. We also identify a novel functional domain within the N-terminal half of FOXP3, which is required for FOXP3-mediated repression of transcription from both a constitutively active and a NF-AT-inducible promoter. Furthermore, we demonstrate that IPEX mutations in these domains correlate with deficiencies in FOXP3 repressor function, corroborating their in vivo relevance.

Effector functions mediated by NK cells involve cytotoxicity and transcription-dependent production and release of cytokines and chemokines. Although the JAK/STAT pathway mediates lymphokine-induced transcriptional regulation in NK cells, very little is known about transcriptional regulation induced during cell-cell contact. We demonstrate that the Wiskott-Aldrich syndrome protein (WASp) is an important component for integration of signals leading to nuclear translocation of NFAT2 and NF-kappaB (RelA) during cell-cell contact and NKp46-dependent signaling. This WASp function is independent of its known role in F-actin polymerization and cytoskeletal rearrangement. Absence of WASp results in decreased accumulation of calcineurin, WASp-interacting protein, and molecules upstream of calcium mobilization, i.e., activated ZAP70 and phospholipase C-gamma1, in the disorganized NK cell immune synapse. Production of GM-CSF, but not IFN-gamma, is decreased, while natural cytotoxicity of Wiskott-Aldrich syndrome-NK cells is maintained. Our results indicate that WASp independently regulates its dual functions, i.e., actin cytoskeletal remodeling and transcription in NK cells.

X-linked agammaglobulinemia (XLA) is a primary immunodeficiency disease caused by mutations of Bruton tyrosine kinase (Btk); Btk plays an essential role in the development of mature B cells. However, small numbers of B cells ("leaky B cells") are present in the peripheral blood of most XLA patients. In this study, we analyzed the function of these leaky B cells obtained from XLA patients. Enough numbers of B cells were available for analysis from five of nine XLA patients originally screened. Sequence analysis revealed missense mutations of Btk in four of the five XLA patients. No mutation was found in the coding region of Btk in one patient. Western blotting and/or flow cytometric analysis failed to detect Btk protein in all five patients. B cells isolated from peripheral blood of these XLA patients were CD5-, CD20+, CD19+, and CD21-. If stimulated with anti-CD40 and IL-4, XLA B cells proliferated normally and produced significant amounts of IgE. Anti-CD40 stimulation of XLA B cells resulted in normal expression of CD23. In addition, three of the five XLA patients studied were immunized with bacteriophage phiX174 and produced low but detectable levels of antiphage-specific Ab. Similarly, X-linked immunodeficiency mice, which carry a missense mutation in Btk, produced substantial amounts of antiphage Ab. These results indicate that CD40 signaling is intact in B cells lacking demonstrable Btk, and that leaky B cells in XLA patients can proliferate, undergo isotype switching, and differentiate into specific Ab-producing cells.

We used Anger camera imaging in a monkey model to investigate the organ localization of a prototype particulate pathogen, 131I-labeled bacteriophage phi X174, after it was bound to the primate erythrocyte complement receptor and then cleared from the circulation. This 131I-labeled phi X174 was infused into the circulation of an immunized monkey, and the nascently formed immune complexes showed rapid and quantitative binding to erythrocytes via the immune adherence reaction (complement-mediated binding). Alternatively, phi X174 was infused into the circulation of a naive animal, and then cross-linked bispecific mAb complexes (heteropolymers, anti-CR1 x anti-phi X174) were infused into the circulation. The infused heteropolymers also facilitated rapid and quantitative binding of phi X174 to erythrocytes. In both cases, after a short lag period, the erythrocyte-bound phi X174 was rapidly cleared from the circulation, and the vast majority of the radiolabel was cleared to the liver, with a small amount clearing to the spleen. Further liver imaging confirmed that within 24 h most of the bacteriophage previously cleared to the liver via the heteropolymer system was phagocytosed and destroyed. The findings in this model system provide additional evidence for the potential utility of heteropolymers to facilitate the safe and rapid clearance of blood-borne pathogens as a potential treatment for infectious diseases.

We investigated the feasibility of using bispecific mAb complexes to redirect and improve the efficiency of the primate E complement receptor 1-based clearance reaction to remove a virus from the circulation. As an initial approach, we used bacteriophage phiX174 as an immunologic model for mammalian viruses. Bispecific complexes were prepared by chemically cross-linking a mAb specific for complement receptor 1 with a mAb specific for the bacteriophage phiX174. In a monkey model these complexes facilitate rapid and quantitative binding of the target bacteriophage to E in vitro and in vivo. Moreover, after in vivo binding to E, the complexes containing mAb and prototype virus are rapidly cleared from the circulation of rhesus and cynomolgus monkeys without loss of E. Our findings suggest that bispecific mAb complexes, in concert with primate E complement receptor 1, may have therapeutic utility in the treatment of diseases associated with blood-borne pathogens.

We report an association between X-linked immunodeficiency with hyper-IgM (XHIM) and carcinomas affecting the liver, pancreas, biliary tree, and associated neuroectodermal endocrine cells. The tumors were fatal in eight of nine cases and in most instances were preceded by chronic cholangiopathy and/or cirrhosis. An additional group of subjects with XHIM had chronic inflammation of the liver or bile ducts but no malignancy. Many patients with XHIM were infected with cryptosporidia. CD40 is normally expressed on regenerating or inflammed bile duct epithelium. A CD40+ hepatocellular carcinoma cell line, HepG2, susceptible to cryptosporidia and CMV infection became resistant when cell surface CD40 was cross-linked by a CD40 ligand fusion protein. Apoptosis was triggered in HepG2 cells if protein synthesis was blocked by cycloheximide or if the cells were infected by cryptosporidia. Ligation of CD40 on biliary epithelium may contribute to defense against infection by intracellular pathogens. We propose that the CD40 ligand mutations that cause XHIM deprive the biliary epithelium of one line of defense against intracellular pathogens and that malignant transformation in the biliary tree follows chronic infection or inflammation. The resulting tumors may then progress without check by an effective immune response. Patients with XHIM who have abnormal liver function tests should be considered at increased risk for cholangiopathy or malignancy.

Changes in T cell helper function were analyzed when anti-CD3-activated T cells were costimulated with mAbs to the CD28 receptor (anti-CD28). T cell-dependent B cell growth and differentiation were consistently augmented if anti-CD3 stimulated-T cells were simultaneously activated with anti-CD28. Although anti-CD28 enhanced IL-2 and IL-4 production, it did not increase B cell responses solely by augmenting production of soluble lymphokines. Anti-CD28 costimulation induced increases on T cells of CD40 ligand (CD40L), known to promote B cell proliferation and Ig secretion. Because anti-CD28 promoted T cell helper functions and expression of CD40L, we examined the dependence for CD40L during T cell-dependent B cell responses. Although soluble CD40 fusion proteins only partially inhibited T cell-dependent B cell activation, we found a strict requirement for CD40L expression at initiating B cell responses. Both CD40L expression and T cell help were blocked by cyclosporin A after TCR cross-linking, and, unlike T cell proliferation, both remained cyclosporin A sensitive during CD28 costimulation. In addition, anti-CD28 could not compensate for the T cell helper deficiency of hyper IgM syndrome patients who lack functional CD40L. Thus, anti-CD28-induced T cell help is delivered via a CD40L-dependent process. The fact that cross-linking CD40 on B cells promotes expression of the B7/BB-1 ligand for CD28 suggest T and B interactions may have a reciprocal amplification mechanism.

To explore the extent of immune reconstitution of SCID mice by human peripheral blood lymphocytes (hu-PBL-SCID mice), we studied the production of immunoglobulin isotypes and specific antibody (Ab) by the engrafted human cells. Human IgG was detectable in 94% of hu-PBL-SCID mice. IgE synthesis by hu-PBL-SCID mice correlated with the IgE levels observed in human donors. All SCID mice receiving PBL obtained from human donors previously immunized with the T-cell-dependent Ag, bacteriophage phi x 174 (phage), produced phage neutralizing antibody. Quantity and quality (Ig isotypes) of phage-specific Ab produced by hu-PBL-SCID mice correlated with that observed in the donor serum. Human B cells alone failed to engraft, and T cells were required for the production of Ig and anti-phage Ab. Phage-specific Ab production occurred without direct Ag exposure of the hu-PBL-SCID mice, suggesting that the specific Ab production was induced directly by polyclonal activation of the engrafted human cells. Intravenous phage injections given 4 wk after cell transfer failed to further increase the anti-phage Ab titer. Phage neutralizing Ab production could not be boosted if spleen cells obtained from hu-PBL-SCID mice were cultured in the presence of Ag. However, hu-PBL-SCID mice produced increased amounts of anti-phage Ab, providing they were injected with phage at the time of cell transfer. Injection of phage at the time of cell transfer, but not 4 wk later, to mice receiving PBL from nonimmunized donors induced production of minute amounts of anti-phage Ab. We conclude that human peripheral blood lymphocytes transferred into SCID mice become maximally stimulated presumably by xenogeneic murine Ag, resulting in polyclonal expansion of the graft and spontaneous production of Ab to Ag the human donor was previously exposed to, and in loss of responses to subsequent Ag exposure. Ab production to neoantigen, however, can be induced and that to recall Ag can be modified if PBL are exposed to Ag at the time of cell transfer.

Mice with immunodeficiency provide an excellent in vivo model for cell transfer experiments. In this study, we compare the extent of immune deficiency of the original CB17 severe combined immune-deficient (SCID) mice with that of two other strains of immune-deficient mice, the recently developed C3H SCID mice and the beige/nude/X-linked immune-deficient (BNX) mice. Detectable levels of serum lg (higher than 0.4 microgram/ml) were found in 79% of CB17 SCID mice studied (n = 24) and in all BNX mice (n = 12); some leaky CB17 SCID mice had normal levels of Ig. In contrast, only 15% of C3H SCID mice (n = 61) had detectable serum lg; the highest Ig level in this strain was 9.6 micrograms/ml. Age had no effect on serum Ig concentrations of C3H SCID mice; in contrast, all old (> 1-year-old) CB17 SCID mice studied had detectable levels of serum Ig. Transfer of syngeneic, normal, neonatal thymocytes increased serum Ig of SCID mouse origin to near-normal levels in all CB17 SCID mice but had no effect on serum lg concentrations in C3H SCID mice. Treatment with anti-asialo-GM-1 antiserum to abrogate NK cell activity increased serum Ig levels in 37% of CB17 SCID mice but had no effect on Ig production in C3H SCID mice. Flow cytometric analysis failed to identify mature T or B cells in C3H SCID mice; in contrast, some leaky CB17 SCID mice had detectable numbers of T and B cells in the peritoneal cavity. After immunization with bacteriophage phi X 174, neither C3H nor CB17 SCID mice, including leaky mice, produced specific antibody to phage. In contrast, BNX mice produced small but significant amounts of anti-phage antibody. These results indicate that, of the three strains of immune-deficient mice, C3H SCID mice have the most severe immune defect. We predict that C3H SCID mice will be best suited for cell transfer experiments.

Previous studies have shown that animals pharmacologically depleted of C3 have impaired antibody responses. However, such C depletion is neither complete nor sustained, and the C3 cleavage products generated by C3 depletion can both enhance and inhibit the immune response. To clarify the role of C3 in humoral immunity, the antibody response of dogs with genetically determined total deficiency of C3 (C3D) was examined. Serum IgG levels of the C3D animals were within the normal range, but were significantly lower than levels seen in normal controls or C3D heterozygotes. Specific antibody production was defective: the antibody titers of C3D dogs in response to primary intravenous immunization with two different T cell-dependent Ag (sheep E and bacteriophage phi X-174) were markedly reduced when compared to either normal controls or C3D heterozygotes. After secondary immunization with T-dependent Ag, the total antibody titers were normal, but the C3D dogs made proportionately more IgM and less IgG antibody than did either control group. After i.v. immunization with a T cell-independent Ag (DNP-Ficoll), the C3D dogs had reduced levels of IgM and IgG antibody after primary and secondary immunization. Neither i.m. immunization nor the use of a 20-fold increase in Ag dose i.v. could correct the defect seen in the antibody response of C3D dogs. The results herein demonstrate that C3 plays a critical role in the generation of a normal humoral immune response.

We have evaluated the binding of human peripheral blood monocytes to cultured vascular endothelium as an in vitro model of monocyte interaction with the vessel wall. Monocytes were purified (91% +/- 4 SE esterase positive) by elutriation to avoid contact with surfaces before assay. Adherence of 51Cr-labeled monocytes after 45 min (36% +/- 11 SE) was significantly higher than that observed with autologous radiolabeled neutrophils (9% +/- 5 SE) and was greater on monolayers of human umbilical vein endothelium than on bovine aortic endothelium. Peripheral blood mononuclear cells treated with monoclonal antibody (MoAb) 60.3, a reagent that binds leukocyte membrane complex CDw18, implicated in multiple adherence-dependent functions, failed to adhere and flatten on artificial surfaces. Mononuclear cells treated with MoAb 60.3 simulated cells from a patient with recurrent infections whose phagocytes failed to react with MoAb 60.3 and failed to emigrate to extravascular sites in vivo. Incubation of monocytes with MoAb 60.3 inhibited (by 32 to 61%) monocyte adherence to endothelium in a dose-dependent manner for periods up to 24 hr, but had negligible effects on basal (unstimulated) neutrophil adherence. Basal monocyte adherence in the presence of MoAb 60.3 remained significantly greater than basal neutrophil adherence. Augmentation of phagocyte adherence to endothelial monolayers by autologous plasma or phorbol ester (PMA) was abrogated by incubation with MoAb 60.3. Studies with immunofluorescence flow cytometry indicated that PMA stimulation of monocytes resulted in a specific 40% increase in monocyte surface expression of the epitope recognized by MoAb 60.3. These in vitro findings, in conjunction with observations from two patients, support the hypothesis that monocyte adherence to endothelium and emigration to tissues is mediated by mechanisms both dependent upon and independent of the CDw18 complex and the epitope recognized by MoAb 60.3.

We examined the role of the neutrophil membrane antigen complex designated CDw18 (LFA-1/Mac-1/p150, 95) in human peripheral blood neutrophil adherence to cultured human umbilical vein endothelial cells (HEC) pretreated with lipopolysaccharide (LPS), interleukin 1 (IL 1), or recombinant tumor necrosis factor-alpha (rTNF-alpha). Pretreatment of HEC with LPS produced a dose-and time-dependent increase in subsequent neutrophil adherence (7 +/- 1% adherence to untreated HEC vs 38 +/- 3% adherence to HEC pretreated for 4 hr with LPS 150 ng/ml; mean +/- SE of 22 experiments: p less than 0.001). This effect was observed in primary and passaged HEC, but not in bovine aortic endothelial cells or human dermal fibroblasts. The LPS-induced activity appeared to be associated with the HEC surface, since it was not removed by washing and was not detected in the supernatant medium. Inhibition of RNA or protein synthesis during pretreatment of HEC with LPS prevented induction of the adherence-promoting activity. Pretreatment of HEC with IL 1 and rTNF-alpha produced a similar protein synthesis-dependent increase in neutrophil adherence to HEC. Coincubation of neutrophils with murine monoclonal antibody (MoAb) 60.3, an antibody directed to the CDw18 complex, produced a 70 +/- 4% inhibition of neutrophil adherence to LPS-pretreated HEC, 59 +/- 5% inhibition of adherence to IL 1-pretreated HEC, and 65 +/- 11% inhibition of adherence to rTNF-alpha-pretreated HEC (means +/- SE of 18, seven, and five experiments, respectively). Notably, MoAb 60.3 did not completely inhibit neutrophil adherence to pretreated HEC, although it completely inhibited adherence to untreated HEC when neutrophils were activated directly with phorbol ester. Similarly, the adherence of neutrophils from a patient with an inherited deficiency of the CDw18 complex to LPS-, IL 1-, and rTNF-alpha-pretreated HEC was markedly reduced compared with normal neutrophils (5 to 11% adherence with CDw18-deficient neutrophils vs 43 to 54% adherence with normal neutrophils), but adherence to pretreated HEC was still significantly greater than adherence to HEC that were not pretreated (2% adherence). We conclude that LPS, IL 1, and rTNF-alpha induce synthesis of an endothelial cell-surface factor(s) that promotes neutrophil adherence primarily by a mechanism involving the CDw18 complex. It thus appears that the CDw18 complex is important for augmented neutrophil adherence to endothelium in vitro whether the is stimulated directly by inflammatory mediators or indirectly by endothelial-dependent mechanisms.

In this study we examined the importance of neutrophil adherence in neutrophil-mediated endothelial cell injury. Phorbol myristate acetate (PMA)-activated neutrophils from a patient with a congenital defect in neutrophil adherence (Gp-150 deficiency) and PMA-activated normal neutrophils pretreated with monoclonal antibody (MoAb) 60.3 were used. Both Gp-150-deficient and MoAb 60.3-treated normal neutrophils failed to adhere to cultured human umbilical vein endothelial cell (HEC) monolayers when activated by PMA (adherence less than 10% with patient and MoAb 60.3-treated cells compared with 53 +/- 3% with normal cells). The addition of PMA-activated normal neutrophils to 51Cr-labeled HEC monolayers failed to induce significant 51Cr release but did produce marked HEC detachment (percentage of detachment 50 +/- 3 at 6 hr). In marked contrast, PMA-activated Gp-150-deficient neutrophils failed to induce significant HEC detachment (percentage of detachment zero (0) at 6 hr). Moreover, the addition of MoAb 60.3 to normal neutrophils inhibited neutrophil-mediated HEC detachment in a time- and dose-dependent fashion. Non-lytic HEC detachment was determined to be largely oxygen radical independent, because PMA-activated chronic granulomatous disease neutrophils and PMA-activated normal neutrophils produced similar disruption of HEC monolayers. Soybean trypsin inhibitor, a chloromethylketone elastase inhibitor, and autologous serum all failed to inhibit neutrophil-mediated HEC detachment. From these studies there is no evidence that nonlytic HEC detachment by PMA-activated neutrophils is mediated by the neutrophil-derived proteases, elastase and cathepsin G. Neutrophil-mediated HEC detachment also required intact neutrophils, because postsecretory medium from PMA-activated normal neutrophils and a suspension of frozen-thawed PMA-activated normal neutrophils were without effect. These in vitro studies indicate that the neutrophil cell surface glycoprotein Gp-150 is required for nonlytic HEC detachment by intact PMA-activated neutrophils.

Two monoclonal antibodies (Mab), designated 60.3 and 60.1, markedly inhibited the phagocytosis of serum-opsonized zymosan by human polymorphonuclear leukocytes (PMN) as measured by the iodination reaction and by microscopic visualization. These antibodies also inhibited rosette formation with EC3bi without decreasing EC3b rosetting, suggesting that Mab 60.3 and 60.1 inhibit the phagocytosis of opsonized zymosan through reaction with the C3bi receptor (CR3) on the leukocyte surface. In support of this concept is the finding that the PMN of two patients with recurrent infections do not ingest opsonized zymosan, lack C3bi receptor function, and react weakly or not at all with Mab 60.3 and 60.1. At concentrations which completely inhibited ingestion of opsonized zymosan, both Mab partially inhibited iodination with Staphylococcus aureus 502A as the particle, and did not affect iodination when Staphylococcus epidermidis was used. This presumably reflects a variable need among the opsonized particles for CR3 for ingestion. Mab 60.3 also inhibited the phagocytosis of certain unopsonized particles as measured by iodination, indicating that the antigens recognized by the Mab do not influence phagocytosis solely by functioning as a C3bi receptor. Mab 60.3 increased the phagocytosis of unopsonized, heat-killed S. aureus by reaction with the PMN via its antibody-combining site, and with the staphylococcal protein A via its Fc region (reverse opsonization). This process required protein A-containing organisms (S. aureus 502A or Cowan 1 but not S. aureus Wood 46 or S. epidermidis), was inhibited by purified protein A, and was not seen either when the F(ab')2 or Fab fragments of the antibody, or when PMN which lack or have low levels of the antigen were employed. Thus, these studies, using iodination as a convenient method for the measurement of phagocytosis, demonstrated two effects of antibodies directed against PMN cell surface components: inhibition of phagocytosis by reaction with the C3bi receptor, and stimulation of phagocytosis by reverse opsonization.