TCR Signaling and CD28/CTLA-4 Signaling Cooperatively Modulate T Regulatory Cell Homeostasis.

Foxp3+ T regulatory cells (Tregs), conventional CD4+Foxp3- T cells, and CD8+ T cells represent heterogeneous populations composed of naive phenotype (NP, CD44low) and memory phenotype (MP, CD44high) subpopulations. NP and MP subsets differ in their activation state, contribution to immune function, and capacity to proliferate in vivo. To further understand the factors that contribute to the differential homeostasis of NP/MP subsets, we examined the differential effects of CD28 and CTLA-4 interaction with CD80/CD86, as well as MHC class II-TCR interaction within mouse Treg pools and CD4+ and CD8+ T cell pools. Blockade of CD80/CD86 with CTLA-4-Ig markedly reduced the cycling and absolute numbers of MP Tregs and MP CD4+ T cells, with minimal effect on the NP T cell subpopulations. Blockade of MHC class II-TCR interaction led to selective expansion of MP Tregs and MP CD4+ and CD8+ T cells that was reversed upon cotreatment with CTLA-4-Ig. Treatment with anti-CTLA-4 mAb altered MP Treg and MP CD4+ and CD8+ T cell homeostasis in a manner similar to that observed with anti-MHC class II. We postulate a complex pathway in which CD28 is the primary driver of Treg proliferation and CTLA-4 functions as the main brake but is likely dependent on TCR signals and CD80/CD86. These findings have important implications for the use of biologic agents targeting such pathways to modulate autoimmune and neoplastic disease.

IFN-α/ß receptor signaling promotes regulatory T cell development and function under stress conditions.

Type I IFNs are a family of cytokines with antiviral and immunomodulatory properties. Although the antiviral effects of IFNs are well characterized, their immunomodulatory properties are less clear. To specifically address the effects of type I IFNs on T regulatory cells (Tregs), we studied mixed bone marrow chimeras between wild-type and IFN-α/ß receptor (IFNAR) knockout (KO) mice, and heterozygous female mice expressing a Treg-specific deletion of the IFNAR. In these two models, IFNAR signaling promotes the development of the Treg lineage in the thymus and their survival in the periphery. IFNAR KO Tregs had a higher expression of the proapoptotic gene Bim and higher frequency of active caspase-positive cells. IFNAR KO Tregs from chimeric mice displayed a more naive phenotype, accompanied by lower levels of CD25 and phosphorylated STAT5. Therefore, in Tregs, IFNAR signaling may directly or indirectly affect phosphorylation of STAT5. In mixed chimeras with Scurfy fetal liver, Tregs derived from IFNAR KO bone marrow were unable to control T effector cell activation and tissue inflammation. Under stress conditions or in a competitive environment, IFNAR signaling may be required to maintain Treg homeostasis and function.

Antibody effector functions mediated by Fcγ-receptors are compromised during persistent viral infection.

T cell dysfunction is well documented during chronic viral infections but little is known about functional abnormalities in humoral immunity. Here we report that mice persistently infected with lymphocytic choriomeningitis virus (LCMV) exhibit a severe defect in Fcγ-receptor (FcγR)-mediated antibody effector functions. Using transgenic mice expressing human CD20, we found that chronic LCMV infection impaired the depletion of B cells with rituximab, an anti-CD20 antibody widely used for the treatment of B cell lymphomas. In addition, FcγR-dependent activation of dendritic cells by agonistic anti-CD40 antibody was compromised in chronically infected mice. These defects were due to viral antigen-antibody complexes and not the chronic infection per se, because FcγR-mediated effector functions were normal in persistently infected mice that lacked LCMV-specific antibodies. Our findings have implications for the therapeutic use of antibodies and suggest that high levels of pre-existing immune complexes could limit the effectiveness of antibody therapy in humans.

Endogenous mouse mammary tumor viruses (mtv): new roles for an old virus in cancer, infection, and immunity.

Mouse Mammary Tumor Viruses are beta-retroviruses that exist in both exogenous (MMTV) and endogenous (Mtv) forms. Exogenous MMTV is transmitted via the milk of lactating animals and is capable of inducing mammary gland tumors later in life. MMTV has provided a number of critical models for studying both viral infection as well as human breast cancer. In addition to the horizontally transmitted MMTV, most inbred mouse strains contain permanently integrated Mtv proviruses within their genome that are remnants of MMTV infection and vertically transmitted. Historically, Mtv have been appreciated for their role in shaping the T cell repertoire during thymic development via negative selection. In addition, more recent work has demonstrated a larger role for Mtv in modulating host immune responses due to its peripheral expression. The influence of Mtv on host response has been observed during experimental murine models of Polyomavirus- and ESb-induced lymphoma as well as Leishmania major and Plasmodium berghei ANKA infection. Decreased susceptibility to bacterial pathogens and virus-induced tumors has been observed among mice lacking all Mtv. We have also demonstrated a role for Mtv Sag in the expansion of regulatory T cells following chronic viral infection. The aim of this review is to summarize the latest research in the field regarding peripheral expression of Mtv with a particular focus on their role and influence on the immune system, infectious disease outcome, and potential involvement in tumor formation.

IL-2 controls the stability of Foxp3 expression in TGF-beta-induced Foxp3+ T cells in vivo.

Stimulation of naive mouse CD4(+)Foxp3(-) T cells in the presence of TGF-ß results in the induction of Foxp3 expression and T suppressor function. However, Foxp3 expression in these induced regulatory T cells (iTreg) is unstable, raising the possibility that iTreg would not be useful for treatment of autoimmune diseases. To analyze the factors that control the stability of Foxp3 expression in iTreg, we generated OVA-specific iTreg from OT-II Foxp3-GFP knockin mice. Following transfer to normal C57BL/6 mice, OT-II GFP(+) cells maintained high levels of Foxp3 expression for 8 d. However, they rapidly lost Foxp3 expression upon stimulation with OVA in IFA in vivo. This unstable phenotype was associated with a strong methylation of the Treg-specific demethylated region within the Foxp3 locus. Administration of IL-2/anti-IL-2 complexes expanded the numbers of transferred Foxp3(+) iTreg in the absence of Ag challenge. Notably, when the iTreg were stimulated with Ag, treatment with IL-2/anti-IL-2 complexes stabilized Foxp3 expression and resulted in enhanced demethylation of the Treg-specific demethylated region. Conversely, neutralization of IL-2 or disruption of its signaling by deletion of Stat5 diminished the level of Foxp3 expression resulting in decreased suppressor function of the iTreg in vivo. Our data suggest that stimulation with TGF-ß in vitro is not sufficient for imprinting T cells with stable expression of Foxp3. Administration of IL-2 in vivo results in stabilization of Foxp3 expression and may prove to be a valuable adjunct for the use of iTreg for the treatment of autoimmune diseases.

Regulatory T-cell expansion during chronic viral infection is dependent on endogenous retroviral superantigens.

Regulatory T cells (Treg) play critical roles in the modulation of immune responses to infectious agents. Further understanding of the factors that control Treg activation and expansion in response to pathogens is needed to manipulate Treg function in acute and chronic infections. Here we show that chronic, but not acute, infection of mice with lymphocytic choriomeningitis virus results in a marked expansion of Foxp3(+) Treg that is dependent on retroviral superantigen (sag) genes encoded in the mouse genome. Sag-dependent Treg expansion was MHC class II dependent, CD4 independent, and required dendritic cells. Thus, one unique mechanism by which certain infectious agents evade host immune responses may be mediated by endogenous Sag-dependent activation and expansion of Treg.

Identification of antigenic Brugia adult worm proteins by peptide mass fingerprinting.

With the recent completion of the Brugia malayi genome, proteomics offers a new resource for a deeper understanding of the biology of filarial parasites. We employed 2-dimensional (2D) gel electrophoresis followed by peptide mass fingerprinting on a matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF) mass spectrometer to identify Brugia adult worm proteins and then determined which proteins were recognized by the host humoral immune response. We identified 18 unique proteins, several of which were determined to be antigenic by immunoblot. The proteins identified here may contribute to future studies to analyze the transmission and pathogenesis of lymphatic filariasis.

TGF-beta-induced Foxp3+ regulatory T cells rescue scurfy mice.

Scurfy mice have a deletion in the forkhead domain of the forkhead transcription factor p3 (Foxp3), fail to develop thymic-derived, naturally occurring Foxp3+ regulatory T cells (nTreg), and develop a fatal lymphoproliferative syndrome with multi-organ inflammation. Transfer of thymic-derived Foxp3+ nTreg into neonatal Scurfy mice prevents the development of disease. Stimulation of conventional CD4+Foxp3(-) via the TCR in the presence of TGF-beta and IL-2 induces the expression of Foxp3 and an anergic/suppressive phenotype. To determine whether the TGF-beta-induced Treg (iTreg) were capable of suppressing disease in the Scurfy mouse, we reconstituted newborn Scurfy mice with polyclonal iTreg. Scurfy mice treated with iTreg do not show any signs of disease and have drastically reduced cell numbers in peripheral lymph nodes and spleen in comparison to untreated Scurfy controls. The iTreg retained their expression of Foxp3 in vivo for 21 days, migrated into the skin, and prevented the development of inflammation in skin, liver and lung. Thus, TGF-beta-differentiated Foxp3+ Treg appear to possess all of the functional properties of thymic-derived nTreg and represent a potent population for the cellular immunotherapy of autoimmune and inflammatory diseases.

Elimination of Onchocercia volvulus transmission in the Santa Rosa focus of Guatemala.

To eliminate transmission of Onchocerca volvulus, semiannual mass treatment with ivermectin (Mectizan; donated by Merck & Co) has been underway in Guatemala since 2000. We applied the 2001 World Health Organization (WHO) elimination criteria in the Santa Rosa focus of onchocerciasis transmission in Guatemala (10,923 persons at risk). No evidence of parasite DNA was found in 2,221 Simulium ochraceum vectors (one-sided 95% confidence interval [CI], 0-0.086%), and no IgG4 antibody positives to recombinant antigen OV16 were found in a sample of 3,232 school children (95% CI, 0-0.009%). We also found no evidence of microfilariae in the anterior segment of the eye in 363 area residents (95% CI, 0-0.08%). Our interpretation of these data, together with historical information, suggest that transmission of O. volvulus is permanently interrupted in Santa Rosa and that ivermectin treatments there can be halted.

Regulation of surface coat exchange by differentiating African trypanosomes.

African trypanosomes (Trypanosoma brucei) have a digenetic lifecycle that alternates between the mammalian bloodstream and the tsetse fly vector. In the bloodstream, replicating long slender parasites transform into non-dividing short stumpy forms. Upon transmission into the fly midgut, short stumpy cells differentiate into actively dividing procyclics. A hallmark of this process is the replacement of the bloodstream-stage surface coat composed of variant surface glycoprotein (VSG) with a new coat composed of procyclin. Pre-existing VSG is shed by a zinc metalloprotease activity (MSP-B) and glycosylphosphatidylinositol-specific phospholipase C (GPI-PLC). We now provide a detailed analysis of the coordinate and inverse regulation of these activities during synchronous differentiation. MSP-B mRNA and protein levels are upregulated during differentiation at the same time as proteolysis whereas GPI-PLC levels decrease. When transcription or translation is inhibited, VSG release is incomplete and a substantial amount of protein stays cell-associated. Both modes of release are still evident under these conditions, but GPI hydrolysis plays a quantitatively minor role during normal differentiation. Nevertheless, GPI biosynthesis shifts early in differentiation from a GPI-PLC sensitive structure to a resistant procyclic-type anchor. Translation inhibition also results in a marked increase in the mRNA levels of both MSP-B and GPI-PLC, consistent with negative regulation by labile protein factors. The relegation of short stumpy surface GPI-PLC to a secondary role in differentiation suggests that it may play a more important role as a virulence factor within the mammalian host.

Removal of Wolbachia from Brugia pahangi is closely linked to worm death and fecundity but does not result in altered lymphatic lesion formation in Mongolian gerbils (Meriones unguiculatus).

Approximately 30 years ago, researchers reported intracellular bacteria in filarial nematodes. These bacteria are relatives of the arthropod symbiont Wolbachia and occur in many filarial nematodes, including Brugia pahangi and Brugia malayi. Wolbachia bacteria have been implicated in a variety of roles, including filaria development and fecundity and the pathogenesis of lymphatic lesions associated with filarial infections. However, the role of the bacteria in worm biology or filarial disease is still not clear. The present experiments support previous data showing that tetracycline eliminates or reduces Wolbachia bacteria in B. pahangi in vivo. The elimination of Wolbachia was closely linked to a reduction in female fecundity and the viability of both sexes, suggesting that the killing of Wolbachia is detrimental to B. pahangi. The gerbils treated with tetracycline showed reduced levels of interleukin-4 (IL-4) and IL-5 mRNA in renal lymph nodes and spleens compared with the levels in B. pahangi-infected gerbils not treated with tetracycline. However, similar findings were noted in B. pahangi-infected gerbils treated with ivermectin, suggesting that the loss of circulating microfilariae, not the reduction of Wolbachia bacteria, was associated with the altered cytokine profile. Despite the change in T-cell cytokines, there was no difference in the sizes of renal lymph nodes isolated from gerbils in each treatment group. Furthermore, the numbers, sizes, or cellular compositions of granulomas examined in the lymphatics or renal lymph nodes did not differ with treatment. These data suggest that Wolbachia may not play a primary role in the formation of lymphatic lesions in gerbils chronically infected with B. pahangi.

Characterization of antibody responses to Wolbachia surface protein in humans with lymphatic filariasis.

Symbiotic Wolbachia organisms of filarial nematodes have received much attention as possible chemotherapy targets and disease-causing organisms. In order to further investigate the association between anti-Wolbachia immune responses and chronic filarial disease in humans, antibody responses to Wolbachia surface protein (WSP) were assayed in serum samples collected from 232 individuals living in Leogane, Haiti, an area where Wuchereria bancrofti infection is endemic, and from 67 North Americans with no history of lymphatic filariasis. As opposed to antifilarial antibody responses, which were largely influenced by the patient's infection status, the prevalence and levels of anti-WSP immunoglobulin G (IgG) antibodies among individuals with lymphedema or hydrocele were significantly greater than those in gender- and infection-matched individuals without disease. In at least one case, the anti-WSP IgG response was coincident with the onset of lymphedema development, and among anti-WSP-positive women with lymphedema, anti-WSP IgG levels were negatively correlated with the duration of lymphedema. The presence of anti-WSP IgG was also associated with the severity of inguinal adenopathy among men with hydrocele. In addition to the presence of anti-WSP antibodies among Haitians, 15 of 67 (22%) serum samples collected from individuals from North America, where filariasis is not endemic, were also positive for anti-WSP antibodies. In comparison to those from Haitians, anti-WSP antibodies from North Americans primarily recognized a distinct region of WSP located within the highly conserved second transmembrane domain. The results of this study demonstrate that anti-WSP antibody responses are associated with the presence of chronic filarial morbidity and not filarial infection status in humans and suggest that WSP should be further studied as a potential trigger for the development of filarial disease.

Baylisascaris procyonis in the metropolitan Atlanta area.

Baylisascaris procyonis, the raccoon roundworm responsible for fatal larva migrans in humans, has long been thought to be absent from many regions in the southeastern United States. During spring 2002, 11 (22%) of 50 raccoons trapped in DeKalb County, Georgia, had B. procyonis infection. The increasing number of cases highlight this emerging zoonotic infection.

The pathogenesis of filarial lymphedema: is it the worm or is it the host?

Our understanding of the pathogenesis of filarial lymphedema, although evolving, is still limited. Recurrent bacterial infections play a major role in the progression of lymphedema to elephantiasis, but the host and parasite factors that trigger disease development are not known. Field studies in Haiti show that lymphedema and host responses to parasite antigens cluster in families, consistent with the hypothesis that host genes influence lymphedema susceptibility. The recent recognition that filarial parasites harbor the endosymbiotic bacteria, Wolbachia, also raises questions about the potential contribution of the inflammatory response to Wolbachia antigens to lymphedema development. In this review, we discuss potential risk factors for lymphedema and try to integrate these in a model of pathogenesis.