Intrinsic p53 Activation Restricts Gammaherpesvirus-Driven Germinal Center B Cell Expansion during Latency Establishment.

Gammaherpesviruses (GHV) are DNA tumor viruses that establish lifelong latent infections in lymphocytes. For viruses such as Epstein-Barr virus (EBV) and murine gammaherpesvirus 68 (MHV68), this is accomplished through a viral gene-expression program that promotes cellular proliferation and differentiation, especially of germinal center (GC) B cells. Intrinsic host mechanisms that control virus-driven cellular expansion are incompletely defined. Using a small-animal model of GHV pathogenesis, we demonstrate in vivo that tumor suppressor p53 is activated specifically in B cells that are latently infected by MHV68. In the absence of p53, the early expansion of MHV68 latency was greatly increased, especially in GC B cells, a cell-type whose proliferation was conversely restricted by p53. We identify the B cell-specific latency gene M2, a viral promoter of GC B cell differentiation, as a viral protein sufficient to elicit a p53-dependent anti-proliferative response caused by Src-family kinase activation. We further demonstrate that EBV-encoded latent membrane protein 1 (LMP1) similarly triggers a p53 response in primary B cells. Our data highlight a model in which GHV latency gene-expression programs that promote B cell proliferation and differentiation to facilitate viral colonization of the host trigger aberrant cellular proliferation that is controlled by p53. IMPORTANCE: Gammaherpesviruses cause lifelong infections of their hosts, commonly referred to as latency, that can lead to cancer. Latency establishment benefits from the functions of viral proteins that augment and amplify B cell activation, proliferation, and differentiation signals. In uninfected cells, off-schedule cellular differentiation would typically trigger anti-proliferative responses by effector proteins known as tumor suppressors. However, tumor suppressor responses to gammaherpesvirus manipulation of cellular processes remain understudied, especially those that occur during latency establishment in a living organism. Here we identify p53, a tumor suppressor commonly mutated in cancer, as a host factor that limits virus-driven B cell proliferation and differentiation, and thus, viral colonization of a host. We demonstrate that p53 activation occurs in response to viral latency proteins that induce B cell activation. This work informs a gap in our understanding of intrinsic cellular defense mechanisms that restrict lifelong GHV infection.

Bhlhe40 limits early IL-10 production from CD4+ T cells during Plasmodium yoelii 17X infection.

The cytokine IL-10 suppresses T-cell-mediated immunity, which is required to control infection with Plasmodium yoelii. Consequently, IL-10 can delay the time needed to resolve this infection, leading to a higher parasite burden. While the pathways that lead to IL-10 production by CD4+ T cells are well defined, much less is known about the mediators that suppress the expression of this potent anti-inflammatory cytokine. Here, we show that the transcription factor basic helix-loop-helix family member e40 (Bhlhe40) contributes to controlling parasite burden in response to P. yoelii infection in mice. Loss of Bhlhe40 expression in mice results in higher Il10 expression, higher peak parasitemia, and a delay in parasite clearance. The observed phenotype was not due to defects in T-cell activation and proliferation or the humoral response. Nor was it due to changes in regulatory T-cell numbers. However, blocking IL-10 signaling reversed the outcome in Bhlhe40-/ - mice, suggesting that excess IL-10 production limits their ability to control the infection properly. In addition to suppressing Il10 expression in CD4+ T cells, Bhlhe40 can promote Ifng expression. Indeed, IFN-γ production by CD4+ T cells isolated from the liver was significantly affected by the loss of Bhlhe40. Lastly, Bhlhe40 deletion in T cells resulted in a phenotype similar to that observed in the Bhlhe40-/ - mice, indicating that Bhlhe40 expression in T cells contributes to the ability of mice to control infection with P. yoelii.

IgM+ and IgM- memory B cells represent heterogeneous populations capable of producing class-switched antibodies and germinal center B cells upon rechallenge with P. yoelii.

Memory B cells (MBCs) are essential for maintaining long-term humoral immunity to infectious organisms, including Plasmodium. MBCs are a heterogeneous population whose function can be dictated by isotype or expression of particular surface proteins. Here, aided by antigen-specific B-cell tetramers, MBC populations were evaluated to discern their phenotype and function in response to infection with a nonlethal strain of P. yoelii. Infection of mice with P. yoelii 17X resulted in 2 predominant MBC populations: somatically hypermutated isotype-switched (IgM- ) and IgM+ MBCs that coexpressed CD73 and CD80 that produced antigen-specific antibodies in response to secondary infection. Rechallenge experiments indicated that IgG-producing cells dominated the recall response over the induction of IgM-secreting cells, with both populations expanding with similar timing during the secondary response. Furthermore, using ZsGreen1 expression as a surrogate for activation-induced cytidine deaminase expression alongside CD73 and CD80 coexpression, ZsGreen1+ CD73+ CD80+ IgM+ , and IgM- MBCs gave rise to plasmablasts that secreted Ag-specific Abs after adoptive transfer and infection with P. yoelii. Moreover, ZsGreen1+ CD73+ CD80+ IgM+ and IgM- MBCs could differentiate into B cells with a germinal center phenotype after adoptive transfer. A third population of B cells (ZsGreen1- CD73- CD80- IgM- ) that is apparent after infection responded poorly to reactivation in vitro and in vivo, indicating that these cells do not represent a canonical population of MBCs. Together these data indicated that MBC function is not defined by immunoglobulin isotype, nor does coexpression of key surface markers limit the potential fate of MBCs after recall.

Towards rainbow portable Cytophone with laser diodes for global disease diagnostics.

In vivo, Cytophone has demonstrated the capability for the early diagnosis of cancer, infection, and cardiovascular disorders through photoacoustic detection of circulating disease markers directly in the bloodstream with an unprecedented 1,000-fold improvement in sensitivity. Nevertheless, a Cytophone with higher specificity and portability is urgently needed. Here, we introduce a novel Cytophone platform that integrates a miniature multispectral laser diode array, time-color coding, and high-speed time-resolved signal processing. Using two-color (808 nm/915 nm) laser diodes, we demonstrated spectral identification of white and red clots, melanoma cells, and hemozoin in malaria-infected erythrocytes against a blood background and artifacts. Data from a Plasmodium yoelii murine model and cultured human P. falciparum were verified in vitro with confocal photothermal and fluorescent microscopy. With these techniques, we detected infected cells within 4 h after invasion, which makes hemozoin promising as a spectrally selective marker at the earliest stages of malaria progression. Along with the findings from our previous application of Cytophone with conventional lasers for the diagnosis of melanoma, bacteremia, sickle anemia, thrombosis, stroke, and abnormal hemoglobin forms, this current finding suggests the potential for the development of a portable rainbow Cytophone with multispectral laser diodes for the identification of these and other diseases.

ICOS Expression Is Required for Maintenance but Not the Formation of Germinal Centers in the Spleen in Response to Plasmodium yoelii Infection.

Inducible T cell costimulator (ICOS) plays a key role in the differentiation and maintenance of follicular helper T (Tfh) cells and, thus, germinal center (GC) formation. Previously, our laboratory showed in a Plasmodium chabaudi infection model that Icos-/- mice were significantly impaired in their ability to form GCs despite persistent infection and, thus, a continued antigen (Ag) load. Here, we show that the resolution of primary infection with Plasmodium yoelii was delayed in Icos-/- mice. This phenotype was associated with a reduction in the accumulation of Tfh-like and GC Tfh cells and an early deficiency in Ag-specific antibody (Ab) production. However, Icos-/- mice could form GCs, although they were less frequent in number than in wild-type (WT) mice. Nonetheless, the Ag-specific Abs from Icos-/- mice lacked signs of affinity maturation, suggesting functional defects associated with these GCs. Eventually, these GC structures dissipated more rapidly in Icos-/- mice than in WT mice. Moreover, the ability of Icos-/- mice to form these GC structures is not reliant on the high Ag loads associated with P. yoelii infections, as GC formation was preserved in Icos-/- mice treated with atovaquone. Finally, mice were unable to form secondary GCs in the absence of ICOS after rechallenge. Overall, these data demonstrate the necessity of ICOS in the maintenance of Tfh cells, the formation and maintenance of sufficient numbers of functioning GCs, and the ability to generate new GC structures after reinfection with P. yoelii.

The spleen: "epicenter" in malaria infection and immunity.

The spleen is a complex secondary lymphoid organ that plays a crucial role in controlling blood-stage infection with Plasmodium parasites. It is tasked with sensing and removing parasitized RBCs, erythropoiesis, the activation and differentiation of adaptive immune cells, and the development of protective immunity, all in the face of an intense inflammatory environment. This paper describes how these processes are regulated following infection and recognizes the gaps in our current knowledge, highlighting recent insights from human infections and mouse models.

ICOS signaling promotes a secondary humoral response after re-challenge with Plasmodium chabaudi chabaudi AS.

The co-stimulatory molecule ICOS is associated with the induction and regulation of T helper cell responses, including the differentiation of follicular helper T (Tfh) cells and the formation and maintenance of memory T cells. However, the role of ICOS signaling in secondary immune responses is largely unexplored. Here we show that memory T cell formation and maintenance are influenced by persistent infection with P. chabaudi chabaudi AS infection, as memory T cell numbers decline in wild-type and Icos-/- mice after drug-clearance. Following drug-clearance Icos-/- mice display a relapsing parasitemia that occurs more frequently and with higher peaks compared to wild-type mice after re-challenge. The secondary immune response in Icos-/- mice is characterized by significant impairment in the expansion of effector cells with a Tfh-like phenotype, which is associated with a diminished and delayed parasite-specific Ab response and the absence of germinal centers. Similarly, the administration of an anti-ICOSL antagonizing antibody to wild-type mice before and after reinfection with P. c. chabaudi AS leads to an early defect in Tfh cell expansion and parasite-specific antibody production, confirming a need for ICOS-ICOSL interactions to promote memory B cell responses. Furthermore, adoptive transfer of central memory T (TCM) cells from wild-type and Icos-/- mice into tcrb-/- mice to directly evaluate the ability of TCM cells to give rise to Tfh cells revealed that TCM cells from wild-type mice acquire a mixed Th1- and Tfh-like phenotype after P. c. chabaudi AS infection. While TCM cells from Icos-/- mice expand and display markers of activation to a similar degree as their WT counterparts, they displayed a reduced capacity to upregulate markers indicative of a Tfh cell phenotype, resulting in a diminished humoral response. Together these findings verify that ICOS signaling in memory T cells plays an integral role in promoting T cell effector responses during secondary infection with P. c. chabaudi AS.

The Immunobiology of the Interleukin-12 Family: Room for Discovery.

The discovery of interleukin (IL)-6 and its receptor subunits provided a foundation to understand the biology of a group of related cytokines: IL-12, IL-23, and IL-27. These family members utilize shared receptors and cytokine subunits and influence the outcome of cancer, infection, and inflammatory diseases. Consequently, many facets of their biology are being therapeutically targeted. Here, we review the landmark discoveries in this field, the combinatorial biology inherent to this family, and how patient datasets have underscored the critical role of these pathways in human disease. We present significant knowledge gaps, including how similar signals from these cytokines can mediate distinct outcomes, and discuss how a better understanding of the biology of the IL-12 family provides new therapeutic opportunities.

NK1.1 Expression Defines a Population of CD4+ Effector T Cells Displaying Th1 and Tfh Cell Properties That Support Early Antibody Production During Plasmodium yoelii Infection.

Early plasmablast induction is a hallmark of Plasmodium infection and is thought to contribute to the control of acute parasite burden. Although long understood to be a T-cell dependent phenomenon, regulation of early plasmablast differentiation, however, is poorly understood. Here, we identify a population of CD4+ T cells that express the innate NK cell marker NK1.1 as an important source of T cell help for early plasmablast and parasite-specific Ab production. Interestingly, NK1.1+ CD4+ T cells arise from conventional, naive NK1.1- CD4+ T cells, and their generation is independent of CD1d but critically reliant on MHC-II. CD4+ T cells that express NK1.1 early after activation produce IFN-γ and IL-21, and express the follicular helper T (Tfh) cell markers ICOS, PD-1 and CXCR5 more frequently than NK1.1- CD4+ T cells. Further analysis of this population revealed that NK1.1+ Tfh-like cells were more regularly complexed with plasmablasts than NK1.1- Tfh-like cells. Ultimately, depletion of NK1.1+ cells impaired class-switched parasite-specific antibody production during early Plasmodium yoelii infection. Together, these data suggest that expression of NK1.1 defines a population of rapidly expanding effector CD4+ T cells that specifically promote plasmablast induction during Plasmodium infection and represent a subset of T cells whose modulation could promote effective vaccine design.

IL-17 Promotes Differentiation of Splenic LSK- Lymphoid Progenitors into B Cells following Plasmodium yoelii Infection.

Lineage-Sca-1+c-Kit- (LSK-) cells are a lymphoid progenitor population that expands in the spleen and preferentially differentiates into mature B cells in response to Plasmodium yoelii infection in mice. Furthermore, LSK- derived B cells can subsequently contribute to the ongoing immune response through the generation of parasite-specific Ab-secreting cells, as well as germinal center and memory B cells. However, the factors that promote their differentiation into B cells in the spleen postinfection are not defined. In this article, we show that LSK- cells produce the cytokine IL-17 in response to Plasmodium infection. Using Il-17ra-/- mice, IL-17R signaling in cells other than LSK- cells was found to support their differentiation into B cells. Moreover, primary splenic stromal cells grown in the presence of IL-17 enhanced the production of CXCL12, a chemokine associated with B cell development in the bone marrow, by a population of IL-17RA-expressing podoplanin+CD31- stromal cells, a profile associated with fibroblastic reticular cells. Subsequent blockade of CXCL12 in vitro reduced differentiation of LSK- cells into B cells, supporting a direct role for this chemokine in this process. Immunofluorescence indicated that podoplanin+ stromal cells in the red pulp were the primary producers of CXCL12 after P. yoelii infection. Furthermore, podoplanin staining on stromal cells was more diffuse, and CXCL12 staining was dramatically reduced in Il-17ra-/- mice postinfection. Together, these results identify a distinct pathway that supports lymphoid development in the spleen during acute Plasmodium infection.

Preclinical photoacoustic models: application for ultrasensitive single cell malaria diagnosis in large vein and artery.

In vivo photoacoustic flow cytometry (PAFC) has demonstrated potential for early diagnosis of deadly diseases through detection of rare circulating tumor cells, pathogens, and clots in nearly the entire blood volume. Before clinical application, this promising diagnostic platform requires verification and optimization using adequate preclinical models. We show here that this can be addressed by examination of large mouse blood vessels which are similar in size, depth and flow velocity to human vessels used in PAFC. Using this model, we verified the capability of PAFC for ultrasensitive, noninvasive, label-free, rapid malaria diagnosis. The time-resolved detection of delayed PA signals from deep vessels provided complete elimination of background from strongly pigmented skin. We discovered that PAFC's sensitivity is higher during examination of infected cells in arteries compared to veins at similar flow rate. Our advanced PAFC platform integrating a 1060 nm laser with tunable pulse rate and width, a wearable probe with a focused transducer, and linear and nonlinear nanobubble-amplified signal processing demonstrated detection of parasitemia at the unprecedented level of 0.00000001% within 20 seconds and the potential to further improve the sensitivity 100-fold in humans, that is approximately 106 times better than in existing malaria tests.

ICOS Co-Stimulation: Friend or Foe?

Over the last 15 years, the inducible T cell co-stimulator (ICOS) has been implicated in various immune outcomes, including the induction and regulation of Th1, Th2, and Th17 immunity. In addition to its role in directing effector T cell differentiation, ICOS has also been consistently linked with the induction of thymus-dependent (TD) antibody (Ab) responses and the germinal center (GC) reaction. ICOS co-stimulation, therefore, appears to play a complex role in dictating the course of adaptive immunity. In this article, we summarize the initial characterization of ICOS and its relationship with the related co-stimulatory molecule CD28. We then address the contribution of ICOS in directing an effector T cell response, and ultimately disease outcome, against various bacterial, viral, and parasitic infections. Next, we assess ICOS in the context of TD Ab responses, connecting ICOS signaling to follicular helper T cell differentiation and its role in the GC reaction. Finally, we address the link between ICOS and human autoimmune disorders and evaluate potential therapies aiming to mitigate disease progression by modulating ICOS signaling.

An Atypical Splenic B Cell Progenitor Population Supports Antibody Production during Plasmodium Infection in Mice.

Hematopoietic stem and progenitor cells (HSPCs) function to replenish the immune cell repertoire under steady-state conditions and in response to inflammation due to infection or stress. Whereas the bone marrow serves as the primary niche for hematopoiesis, extramedullary mobilization and differentiation of HSPCs occur in the spleen during acute Plasmodium infection, a critical step in the host immune response. In this study, we identified an atypical HSPC population in the spleen of C57BL/6 mice, with a lineage(-)Sca-1(+)c-Kit(-) (LSK(-)) phenotype that proliferates in response to infection with nonlethal Plasmodium yoelii 17X. Infection-derived LSK(-) cells upon transfer into naive congenic mice were found to differentiate predominantly into mature follicular B cells. However, when transferred into infection-matched hosts, infection-derived LSK(-) cells gave rise to B cells capable of entering into a germinal center reaction, and they developed into memory B cells and Ab-secreting cells that were capable of producing parasite-specific Abs. Differentiation of LSK(-) cells into B cells in vitro was enhanced in the presence of parasitized RBC lysate, suggesting that LSK(-) cells expand and differentiate in direct response to the parasite. However, the ability of LSK(-) cells to differentiate into B cells was not dependent on MyD88, as myd88(-/-) LSK(-) cell expansion and differentiation remained unaffected after Plasmodium infection. Collectively, these data identify a population of atypical lymphoid progenitors that differentiate into B lymphocytes in the spleen and are capable of contributing to the ongoing humoral immune response against Plasmodium infection.

In vivo photoacoustic flow cytometry for early malaria diagnosis.

In vivo photoacoustic (PA) flow cytometry (PAFC) has already demonstrated a great potential for the diagnosis of deadly diseases through ultrasensitive detection of rare disease-associated circulating markers in whole blood volume. Here, we demonstrate the first application of this powerful technique for early diagnosis of malaria through label-free detection of malaria parasite-produced hemozoin in infected red blood cells (iRBCs) as high-contrast PA agent. The existing malaria tests using blood smears can detect the disease at 0.001-0.1% of parasitemia. On the contrary, linear PAFC showed a potential for noninvasive malaria diagnosis at an extremely low level of parasitemia of 0.0000001%, which is ∼10(3) times better than the existing tests. Multicolor time-of-flight PAFC with high-pulse repetition rate lasers at wavelengths of 532, 671, and 820 nm demonstrated rapid spectral and spatial identification and quantitative enumeration of individual iRBCs. Integration of PAFC with fluorescence flow cytometry (FFC) provided real-time simultaneous detection of single iRBCs and parasites expressing green fluorescence proteins, respectively. A combination of linear and nonlinear nanobubble-based multicolor PAFC showed capability to real-time control therapy efficiency by counting of iRBCs before, during, and after treatment. Our results suggest that high-sensitivity, high-resolution ultrafast PAFC-FFC platform represents a powerful research tool to provide the insight on malaria progression through dynamic study of parasite-cell interactions directly in bloodstream, whereas portable hand-worn PAFC device could be broadly used in humans for early malaria diagnosis. © 2016 International Society for Advancement of Cytometry.

The Costimulatory Molecule ICOS Regulates Host Th1 and Follicular Th Cell Differentiation in Response to Plasmodium chabaudi chabaudi AS Infection.

Blood-stage Plasmodium chabaudi chabaudi AS infection requires cell- and Ab-mediated immunity to control acute and persistent infection, respectively. ICOS regulates CD4(+) T cell activation and promotes the induction of follicular Th (TFH) cells, CD4(+) T cells that support B cell affinity maturation within germinal centers (GCs), resulting in the production of high-affinity Abs. In this article, we demonstrate that, in response to P. c. chabaudi AS infection, the absence of ICOS resulted in an enhanced Th1 immune response that reduced peak parasitemia. Despite the absence of ICOS, CD4(+) T cells were capable of expressing PD-1, B cell lymphoma 6, and CXCR5 during early infection, indicating TFH development was not impaired. However, by day 21 postinfection, Icos(-/-) mice accumulated fewer splenic TFHs compared with Icos(+/+) mice, leading to substantially fewer GC B cells and a decrease in affinity, but not production, of parasite-specific isotype-switched Abs. Moreover, treatment of mice with anti-ICOS ligand Abs to modulate ICOS-ICOS ligand signaling revealed a requirement for ICOS in TFH differentiation only after day 6 postinfection. Ultimately, the quality and quantity of isotype-switched Abs produced in Icos(-/-) mice declined over time, resulting in impaired control of persistent parasitemia. Collectively, these data suggest ICOS is not required for TFH induction during P. c. chabaudi AS infection or production of isotype-switched Abs, but it is necessary for maintenance of a sustained high-affinity, protective Ab response.

Timed action of IL-27 protects from immunopathology while preserving defense in influenza.

Infection with influenza virus can result in massive pulmonary infiltration and potentially fatal immunopathology. Understanding the endogenous mechanisms that control immunopathology could provide a key to novel adjunct therapies for this disease. Here we show that the cytokine IL-27 plays a crucial role in protection from exaggerated inflammation during influenza virus infection. Using Il-27ra-/- mice, IL-27 was found to limit immunopathology, neutrophil accumulation, and dampened TH1 or TH17 responses via IL-10-dependent and -independent pathways. Accordingly, the absence of IL-27 signals resulted in a more severe disease course and in diminished survival without impacting viral loads. Consistent with the delayed expression of endogenous Il-27p28 during influenza, systemic treatment with recombinant IL-27 starting at the peak of virus load resulted in a major amelioration of lung pathology, strongly reduced leukocyte infiltration and improved survival without affecting viral clearance. In contrast, early application of IL-27 impaired virus clearance and worsened disease. These findings demonstrate the importance of IL-27 for the physiological control of immunopathology and the potential value of well-timed IL-27 application to treat life-threatening inflammation during lung infection.

IL-21 is required for optimal antibody production and T cell responses during chronic Toxoplasma gondii infection.

Previous studies have indicated that Il21r (-/-) mice chronically infected with Toxoplasma gondii display a defect in serum IgG; however, the basis for this antibody defect was not defined and questions remain about the role of IL-21 in promoting the production of IL-10, which is required to limit infection-induced pathology during toxoplasmosis. Therefore, Il21 (-/-) mice were challenged with T. gondii to determine whether IL-21 impacts the parasite-specific CD8(+) T cell response, its contribution to thymus-dependent antibody production after infection, and balance between protective and pathogenic responses. Whereas IL-21 has been implicated in the differentiation of IL-10 producing CD4(+) T cells no immune-mediated pathology was evident in Il21 (-/-) mice during the acute response, nor was there a defect in the development of this population in chronically infected Il21 (-/-) mice. However, Il21 (-/-) mice displayed a defect in IgG production after infection that correlated with a decrease in GC B cell numbers, the CD4(+) and CD8(+) T cell numbers in the brain were reduced over the course of the chronic infection leading to a decrease in total IFN-γ production and an increase in parasite numbers associated with susceptibility to toxoplasmic encephalitis. Together, these results identify a key role for IL-21 in shaping the humoral and cellular response to T. gondii, but indicate that IL-21 has a limited role in regulating immunopathology.

Do you see what I see: Recognition of protozoan parasites by Toll-like receptors.

Toll-like receptors (TLRs) are important for recognizing a variety of pathogens, including protozoan parasites, and initiating innate immune responses against them. TLRs are localized on the cell surface as well as in the endosome, and are implicated in innate sensing of these parasites. In this review, we will discuss recent findings on the identification of parasite-derived pathogen associated molecular patterns and the TLRs that bind them. The role of these TLRs in initiating the immune response against protozoan parasitic infections in vivo will be presented in the context of murine models of infection utilizing TLR-deficient mice. Additionally, we will explore evidence that TLRs and genetic variants of TLRs may impact the outcome of these parasitic infections in humans.

Parasites: what are they good for?

Parasitic diseases caused by helminth and protozoan infections remain one of the largest global public health problems for mankind. While natural immunity in man is rare or slow to develop for many parasites, the immune response is capable of recognizing and responding to infection by utilizing a number of different immunological mechanisms. This special topics journal issue examines many of the key findings in the recent literature regarding the immune response against helminth and protozoan infections, as well as highlighting areas in which our current knowledge falls short. The question of how we can tailor immune responses to prevent or reduce disease burden is a burning question within the field of immunoparasitology.

The cytokines interleukin 27 and interferon-γ promote distinct Treg cell populations required to limit infection-induced pathology.

Interferon-γ (IFN-γ) promotes a population of T-bet(+) CXCR3(+) regulatory T (Treg) cells that limit T helper 1 (Th1) cell-mediated pathology. Our studies demonstrate that interleukin-27 (IL-27) also promoted expression of T-bet and CXCR3 in Treg cells. During infection with Toxoplasma gondii, a similar population emerged that limited T cell responses and was dependent on IFN-γ in the periphery but on IL-27 at mucosal sites. Transfer of Treg cells ameliorated the infection-induced pathology observed in Il27(-/-) mice, and this was dependent on their ability to produce IL-10. Microarray analysis revealed that Treg cells exposed to either IFN-γ or IL-27 have distinct transcriptional profiles. Thus, IFN-γ and IL-27 have different roles in Treg cell biology and IL-27 is a key cytokine that promotes the development of Treg cells specialized to control Th1 cell-mediated immunity at local sites of inflammation.