Regulation of the Interleukin (IL)-12R β2 Subunit Expression in Developing T Helper 1 (Th1) and Th2 Cells

The developmental commitment to a T helper 1 (Th1)- or Th2-type response can significantly influence host immunity to pathogens. Extinction of the IL-12 signaling pathway during early Th2 development provides a mechanism that allows stable phenotype commitment. In this report we demonstrate that extinction of IL-12 signaling in early Th2 cells results from a selective loss of IL-12 receptor (IL-12R) β2 subunit expression. To determine the basis for this selective loss, we examined IL-12R β2 subunit expression during Th cell development in response to T cell treatment with different cytokines. IL-12R β2 is not expressed by naive resting CD4⁺ T cells, but is induced upon antigen activation through the T cell receptor. Importantly, IL-4 and IFN-γ were found to significantly modify IL-12 receptor β2 expression after T cell activation. IL-4 inhibited IL-12R β2 expression leading to the loss of IL-12 signaling, providing an important point of regulation to promote commitment to the Th2 pathway. IFN-γ treatment of early developing Th2 cells maintained IL-12R β2 expression and restored the ability of these cells to functionally respond to IL-12, but did not directly inhibit IL-4 or induce IFN-γ production. Thus, IFN-γ may prevent early Th cells from premature commitment to the Th2 pathway. Controlling the expression of the IL-12R β2 subunit could be an important therapeutic target for the redirection of ongoing Th cell responses.     

Interferon γ Derived from CD4+ T Cells Is Sufficient to Mediate T Helper Cell Type 1 Development

Interferon γ (IFN-γ) has been implicated in T helper type 1 (Th1) cell development through its ability to optimize interleukin 12 (IL-12) production from macrophages and IL-12 receptor expression on activated T cells. Various systems have suggested a role for IFN-γ derived from the innate immune system, particularly natural killer (NK) cells, in mediating Th1 differentiation in vivo. We tested this requirement by reconstituting T cell and IFN-γ doubly deficient mice with wild-type CD4⁺ T cells and challenging the mice with pathogens that elicited either minimal or robust IL-12 in vivo (Leishmania major or Listeria monocytogenes, respectively). Th1 cells developed under both conditions, and this was unaffected by the presence or absence of IFN-γ in non-T cells. Reconstitution with IFN-γ–deficient CD4⁺ T cells could not reestablish control over L. major, even in the presence of IFN-γ from the NK compartment. These data demonstrate that activated T cells can maintain responsiveness to IL-12 through elaboration of endogenous IFN-γ without requirement for an exogenous source of this cytokine.     

Single Cell Analysis Reveals Regulated Hierarchical T Cell Antigen Receptor Signaling Thresholds and Intraclonal Heterogeneity for Individual Cytokine Responses of CD4+ T Cells

T cell receptor (TCR) recognition of peptide–major histocompatibility complex antigens can elicit a diverse array of effector activities. Here we simultaneously analyze TCR engagement and the production of multiple cytokines by individual cells in a clonal Th1 CD4⁺ cell population. Low concentrations of TCR ligand elicit only interferon-γ (IFN-γ) production. Increasing ligand recruits more cells into the IFN-γ⁺ pool, increases IFN-γ produced per cell, and also elicits IL-2, but only from cells already making IFN-γ. Most cells producing only IFN-γ show less TCR downmodulation than cells producing both cytokines, consistent with a requirement for more TCR signaling to elicit IL-2 than to evoke IFN-γ synthesis. These studies emphasize the hierarchical organization of TCR signaling thresholds for induction of distinct cytokine responses, and demonstrate that this threshold phenomenon applies to individual cells. The existence of such thresholds suggests that antigen dose may dictate not only the extent, but also the quality of an immune response, by altering the ratios of the cytokines produced by activated T cells. The quantitative relationships in this response hierarchy change in response to costimulation through CD28 or LFA-1, as well as the differentiation state of the lymphocyte, explaining how variations in these parameters in the face of a fixed antigen load can qualitatively influence immune outcomes. Finally, although the IFN-γ/IL-2 hierarchy is seen with most cells, among cells with the greatest TCR downmodulation, some produce only IFN-γ and not IL-2, and the amount of IFN-γ exceeds that in double producers. Thus, these single cell analyses also provide clear evidence of nonquantitative intraclonal heterogeneity in cytokine production by long-term Th1 cells, indicating additional complexity of T cell function during immune responses.     

IL-10 production differentially influences the magnitude,quality, and protective capacity of Th1 responses depending on the vaccine platform

The quality of a Th1 response can be a prospective correlate of vaccine-mediated protection against certain intracellular pathogens. Using two distinct vaccine platforms, we evaluate the influence of interleukin (IL) 10 production on the magnitude, quality, and protective capacity of CD4⁺ T cell responses in the mouse model of Leishmania major infection. Multiparameter flow cytometry was used to delineate the CD4⁺ T cell production of interferon (IFN) γ, IL-2, tumor necrosis factor (TNF), and IL-10 (or combinations thereof) after vaccination. Immunization with a high dose of adenovirus (ADV) expressing leishmanial proteins (MML-ADV) elicited a limited proportion of multifunctional IFN-γ⁺IL-2⁺TNF⁺ Th1 cells, a high frequency of IL-10–producing CD4⁺ T cells, and did not protect against subsequent challenge. Surprisingly, in the absence of IL-10, there was no change in the magnitude, quality, or protective capacity of the Th1 response elicited by high-dose MML-ADV. In contrast, after immunization with MML protein and CpG (MML + CpG), IL-10 limited the production of IL-12 by DCs in vivo, thereby decreasing the generation of multifunctional Th1 cells. Consequently, three immunizations with MML + CpG were required for full protection. However, inhibiting IL-10 at the time of immunization enhanced the magnitude and quality of the Th1 response sufficiently to mediate protection after only a single immunization. Overall, we delineate distinct mechanisms by which vaccines elicit protective Th1 responses and underscore the importance of multifunctional CD4⁺ T cells.CD4⁺ T cell responses after vaccination or infection show substantial heterogeneity in terms of their phenotype and functional capacity. Inducing protective responses without the appropriate means to assess such heterogeneity has limited the ability to define correlates of protection after vaccination. Thus, understanding the type of CD4⁺ T cell response required to mediate protection is critical for rational design of vaccines against infections requiring Th1 immunity. Historically, T cell responses have been characterized by magnitude (frequency), proliferative capacity, or the mean TCR avidity. Recently, multicolor flow cytometry has broadened the spectrum of parameters that can be measured simultaneously at the single-cell level to include phenotypic markers and/or specific combinations of functional responses (e.g., cytokines). This more extensive characterization, termed quality, is defined by the pattern of cytokine production at the single-cell level (Seder et al., 2008). The quality can be related to the spectrum of Th1 differentiation from IFN-γ–negative, IL-2–producing, and/or TNF-producing central memory cells to multifunctional IFN-γ⁺IL-2⁺TNF⁺ or IFN-γ⁺IL-2⁻TNF⁺ effector memory cells to terminally differentiated IFN-γ single-positive cells (Seder et al., 2008; Wu et al., 2002). Furthermore, when quantified on a per-cell basis, Th1 cells secreting all three cytokines (IFN-γ⁺IL-2⁺TNF⁺) produce considerably more IFN-γ than double- or single-producing IFN-γ cells (Darrah et al., 2007). In addition, the ability of multifunctional Th1 cells to also secrete TNF and IL-2 provides additional effector function and enhanced proliferative capacity, respectively, making these cells optimized for durable effector function. The quality of a vaccine-elicited response was first shown to be predictive of disease protection against Leishmania major in as much as a CD4⁺ T cell quality comprising a high frequency of multifunctional IFN-γ⁺IL-2⁺TNF⁺ Th1 cells correlated with protection (Darrah et al., 2007). Moreover, this Th1-based metric has since been correlated with a favorable outcome to a variety of other infections including tuberculosis (Forbes et al., 2008; Lindenstrøm et al., 2009), malaria (Roestenberg et al., 2009), and vaccinia (Trumpfheller et al., 2008).As the quality of a Th1 response may predict outcome against infection, understanding the mechanisms that influence the generation of multifunctional Th1 cells can be used to improve vaccine design. Using various vaccine formulations or by altering the dose of a specific vaccine, we were able to elicit qualitatively distinct Th1 responses that confer varying levels of protection. Indeed, vaccination with a single low dose of MML-adenovirus (ADV) elicited a Th1 response comprising a high frequency of multifunctional cells and protection against L. major challenge, whereas a single high dose of MML-ADV elicited fewer multifunctional cells, a high proportion of IFN-γ single-positive cells, and no protection (Darrah et al., 2007). Although the lack of protection after high-dose MML-ADV immunization was consistent with a poor quality Th1 response, it remained possible that inhibitory cytokines, which have a well established role in limiting protection against L. major (Sacks and Anderson, 2004), were influencing outcome. In this regard, IL-10 produced by CD4⁺ T cells promotes susceptibility and prevents healing in mice and humans infected with L. major (Sacks and Anderson, 2004). In the context of a self-healing L. major infection, IL-10 from natural regulatory T (T reg) cells prevents the eradication of parasites, allowing a persistence of low-level antigen that sustains T cell memory (Belkaid et al., 2001, 2002). Additionally, immune suppression during chronic L. major infection is mediated by IL-10 production from IFN-γ–producing Th1 cells (Anderson et al., 2007). Thus, in the setting of L. major infection in vivo, CD4⁺ T cell–derived IL-10 prevents clearance of parasites. Alternatively, IL-10 produced by Th1 cells can have an important regulatory effect by limiting excess inflammation during Toxoplasma gondii or Flu infection (Jankovic et al., 2002; Sun et al., 2009). Although these studies show that CD4⁺ T cell–derived IL-10 can have distinct regulatory effects on an ongoing infection in vivo, it has not been shown whether CD4⁺IFN-γ⁺IL-10⁺ Th1 cells can be elicited by vaccination with clinically based vectors or how they would influence the quality of the Th1 response or protection. Thus, a major focus of this study was to investigate a role for CD4⁺ T cell–derived (adaptive) IL-10 after immunization with high-dose MML-ADV vaccination and to determine its effect on Th1 quality and protection.IL-10 can also limit Th1 responses indirectly by decreasing APC function or APC production of the canonical polarizing cytokine IL-12 (Moore et al., 2001; Trinchieri, 2001). Indeed, APC-derived (innate) IL-10 induced by vaccination could profoundly impact the efficiency and extent of Th1 differentiation by regulating production of IL-12 by DCs. In this regard, we previously showed three immunizations with MML protein and the toll-like receptor (TLR) 9 ligand CpG (MML + CpG) are required to elicit a high frequency of multifunctional Th1 cells and protective immunity against L. major (Darrah et al., 2007). As CpG is a potent inducer of IL-10 and IL-12 from DCs (Chu et al., 1997; Roman et al., 1997; Boonstra et al., 2006; Samarasinghe et al., 2006), we hypothesized that this innate cross-regulation after immunization might influence the number of immunizations required to achieve protection and the type of Th1 response induced. Thus, the other major focus of the study was to examine the role IL-10 and IL-12 production from innate immune cells on Th1 immunity and protection after MML + CpG immunization.In this study, we investigate how IL-10 production by CD4⁺ T cells or APCs might influence the magnitude, quality, and protective capacity of Th1 responses elicited by MML-ADV or MML + CpG immunization. Thus, a novel multiparameter flow cytometry panel was developed to define the quality of the MML-specific response after vaccination, measured by the production of IL-10, IFN-γ, IL-2, and TNF. In addition, IL-10^−/− mice and WT mice treated with anti–IL-10 receptor (α-IL-10R) were used to define the role of innate and adaptive IL-10 on vaccine-induced immunity and Th1-mediated protection. Overall, this study delineates the mechanisms of how two distinct vaccine formulations generate protective immunity and elucidates the role that IL-10 has on this process.     

NK-DC crosstalk controls the autopathogenic Th17 response through an innate IFN-γ–IL-27 axis

IFN-γ is a pathogenic cytokine involved in inflammation. Paradoxically, its deficiency exacerbates experimental autoimmune encephalomyelitis, uveitis, and arthritis. Here, we demonstrate using IFN-γ^−/− mice repleted with IFN-γ^+/+ NK cells that innate production of IFN-γ from NK cells is necessary and sufficient to trigger an endogenous regulatory circuit that limits autoimmunity. After immunization, DCs recruited IFN-γ-producing NK cells to the draining lymph node and interacted with them in a CXCR3-dependent fashion. The interaction caused DCs to produce IL-27, which in turn enhanced IFN-γ production by NK cells, forming a self-amplifying positive feedback loop. IL-10, produced by the interacting cells themselves, was able to limit this process. The NK-DC–dependent IL-27 inhibited development of the adaptive pathogenic IL-17 response and induced IL-10–producing Tr1-like cells, which ameliorated disease in an IL-10-dependent manner. Our data reveal that an early NK-DC interaction controls the adaptive Th17 response and limits tissue-specific autoimmunity through an innate IFN-γ–IL-27 axis.Autoreactive CD4⁺ T cells have been linked to the pathogenesis of several tissue-specific autoimmune diseases, including multiple sclerosis, arthritis and uveitis. Until recently, Th1 cells and their signature cytokine, IFN-γ, were thought to be the main pathogenic mediators in these types of diseases because (i) autoimmune Th1 cells transfer disease in different animal models; (ii) the IFN-γ level is associated with disease severity in experimental models, including experimental autoimmune uveitis (EAU) and experimental autoimmune encephalomyelitis (EAE; Luger et al., 2008; Haak et al., 2009; Axtell et al., 2010), as well as in human diseases (Link, 1998; Takase et al., 2006); and (iii) transgenic expression of interferon (IFN)-γ in the tissue precipitates inflammation and causes pathology in various organs, directly linking IFN-γ to pathology (Geiger et al., 1994; Horwitz et al., 1997; Link, 1998; Egwuagu et al., 1999; Takase et al., 2006).However, the notion that IFN-γ is solely a pathogenic cytokine is challenged by multiple studies demonstrating that interference with IFN-γ signaling, either by genetic deficiency or by antibodies that target IFN-γ or its receptor, exacerbates disease development (Caspi et al., 1994; Ferber et al., 1996; Jones et al., 1997; Matthys et al., 1998). The discovery that Th17 cells are dominant contributors to tissue damage in the models listed above, and that the development of these effector T cells may be hindered by IFN-γ, led some to propose that IFN-γ was protective because it inhibited the (even more pathogenic) Th17 response.EAU is induced in mice by immunization with the retinal autoantigen interphotoreceptor retinoid-binding protein (IRBP) in CFA and represents an accepted rodent model of human autoimmune uveitis. It is one of a group of autoimmune disease models in which both the protective and the pathogenic effects of IFN-γ can be observed. On the one hand, IFN-γ–producing Th1 cells adoptively transfer disease and locally produced IFN-γ elicits inflammatory pathology in retinal tissue (Geiger et al., 1994; Egwuagu et al., 1999). On the other hand, however, EAU-prone IFN-γ^−/− (GKO) mice exhibit exacerbated disease accompanied by an elevated Th17 response (Jones et al., 1997; Luger et al., 2008). In the course of studies designed to unravel the pathogenic from protective effects of IFN-γ, we noted that acute elicitation of IFN-γ by systemic injections of IL-12 or by stimulation of iNKT cells at the time of uveitogenic immunization, inhibits EAU pathology and blunts development of adaptive immunity (Th17 as well as Th1) to IRBP (Tarrant et al., 1999; Grajewski et al., 2008). Only IFN-γ produced during the first week after disease induction, but not later, had an ameliorating effect. These observations suggested that the protective IFN-γ could be derived from cells within the early/innate, rather than the late/adaptive, compartment. However, the cellular source of IFN-γ that limits autoimmunity in the absence of acute pharmacological stimuli has not been identified.Natural killer (NK) cells are innate effector cells that kill target cells and produce proinflammatory cytokines, including IFN-γ. Recent studies indicate that NK cells can also affect adaptive immunity. In vitro studies documented that interaction between NK cells and DCs leads to activation and maturation of both types of cells and in production of cytokines, including IFN-γ and TNF by NK cells and IL-12, 15 and 18 by DC (Walzer et al., 2005; Deguine and Bousso, 2013). In vivo, NK cells can modulate T cell priming involving antigen-bearing DCs by providing an early source of IFN-γ that promotes the Th1 effector response (Martín-Fontecha et al., 2004). We hypothesized that innate IFN-γ produced by NK cells might explain the IFN-γ-dependent inhibitory effects on development of the adaptive effector response in uveitis, thus explaining the ameliorating effects of early systemic elicitation of IFN-γ on disease.Similar to other autoimmune disease models induced by immunization with a tissue antigen in CFA, EAU is strongly Th17 driven, and IFN-γ knockout (GKO) mice develop exacerbated disease (Luger et al., 2008). By using GKO mice repleted with IFN-γ–sufficient NK cells, we show that IFN-γ from NK cells is necessary to ameliorate disease development and to suppress the adaptive Th17 response, and that IFN-γ derived only from NK cells is sufficient for this activity. Our data show that after uveitogenic immunization, both NK cells and DCs are recruited to the draining LNs and that recruitment of NK cells is dependent on DCs. IFN-γ from NK cells rescues the production of CXCR3 ligands by DCs in GKO mice and expression of CXCR3 by NK cells is important for them to migrate to the draining LNs. Intravital two-photon microscopy revealed that NK cells interact with DCs in the LN in a CXCR3-dependent manner. Mechanistic studies show that this interaction results in production of IFN-γ by NK cells and IL-27 by DCs, with each cytokine promoting production of each other in a positive feedback loop, resulting in inhibited development of adaptive Th17 cells and enhanced development of IL-10–producing T regulatory type 1 (Tr1)-like cells. Our data thus identify an innate IFN-γ–IL-27 axis brought about by NK–DC interaction, which acts to control autoimmunity.     

Ligation of TLR9 induced on human IL-10–secreting Tregs by 1α,25-dihydroxyvitamin D3 abrogates regulatory function

Signaling through the TLR family of molecular pattern recognition receptors has been implicated in the induction of innate and adaptive immune responses. A role for TLR signaling in the maintenance and/or regulation of Treg function has been proposed, however its functional relevance remains unclear. Here we have shown that TLR9 is highly expressed by human Treg secreting the antiinflammatory cytokine IL-10 induced following stimulation of blood and tissue CD3⁺ T cells in the presence of 1α,25-dihydroxyvitamin D3 (1α25VitD3), the active form of Vitamin D, with or without the glucocorticoid dexamethasone. By contrast, TLR9 was not highly expressed by naturally occurring CD4⁺CD25⁺ Treg or by Th1 and Th2 effector cells. Induction of TLR9, but not other TLRs, was IL-10 dependent and primarily regulated by 1α25VitD3 in vitro. Furthermore, ingestion of calcitriol (1α25VitD3) by human volunteers led to an increase of both IL-10 and TLR9 expression by CD3⁺CD4⁺ T cells analyzed directly ex vivo. Stimulation of 1α25VitD3-induced IL-10–secreting Treg with TLR9 agonists, CpG oligonucleotides, resulted in decreased IL-10 and IFN-γ synthesis and a concurrent loss of regulatory function, but, unexpectedly, increased IL-4 synthesis. We therefore suggest that TLR9 could be used to monitor and potentially modulate the function of 1α25VitD3-induced IL-10–secreting Treg in vivo, and that this has implications in cancer therapy and vaccine design.     

IL-17 produced by neutrophils regulates IFN-γ–mediated neutrophil migration in mouse kidney ischemia-reperfusion injury

The IL-23/IL-17 and IL-12/IFN-γ cytokine pathways have a role in chronic autoimmunity, which is considered mainly a dysfunction of adaptive immunity. The extent to which they contribute to innate immunity is, however, unknown. We used a mouse model of acute kidney ischemia-reperfusion injury (IRI) to test the hypothesis that early production of IL-23 and IL-12 following IRI activates downstream IL-17 and IFN-γ signaling pathways and promotes kidney inflammation. Deficiency in IL-23, IL-17A, or IL-17 receptor (IL-17R) and mAb neutralization of CXCR2, the p19 subunit of IL-23, or IL-17A attenuated neutrophil infiltration in acute kidney IRI in mice. We further demonstrate that IL-17A produced by GR-1⁺ neutrophils was critical for kidney IRI in mice. Activation of the IL-12/IFN-γ pathway and NKT cells by administering α-galactosylceramide–primed bone marrow–derived DCs increased IFN-γ production following moderate IRI in WT mice but did not exacerbate injury or enhance IFN-γ production in either Il17a^–/– or Il17r^–/– mice, which suggested that IL-17 signaling was proximal to IFN-γ signaling. This was confirmed by the finding that IFN-γ administration reversed the protection seen in Il17a^–/– mice subjected to IRI, whereas IL-17A failed to reverse protection in Ifng^–/– mice. These results demonstrate that the innate immune component of kidney IRI requires dual activation of the IL-12/IFN-γ and IL-23/IL-17 signaling pathways and that neutrophil production of IL-17A is upstream of IL-12/IFN-γ. These mechanisms might contribute to reperfusion injury in other organs.     

Engagement of Cytotoxic T Lymphocyte–associated Antigen 4 (CTLA-4) Induces Transforming Growth Factor β (TGF-β) Production by Murine CD4+ T Cells

Evidence indicates that cytotoxic T lymphocyte–associated antigen 4 (CTLA-4) may negatively regulate T cell activation, but the basis for the inhibitory effect remains unknown. We report here that cross-linking of CTLA-4 induces transforming growth factor β (TGF-β) production by murine CD4⁺ T cells. CD4⁺ T helper type 1 (Th1), Th2, and Th0 clones all secrete TGF-β after antibody cross-linking of CTLA-4, indicating that induction of TGF-β by CTLA-4 signaling represents a ubiquitous feature of murine CD4⁺ T cells. Stimulation of the CD3–T cell antigen receptor complex does not independently induce TGF-β, but is required for optimal CTLA-4–mediated TGF-β production. The consequences of cross-linking of CTLA-4, together with CD3 and CD28, include inhibition of T cell proliferation and interleukin (IL)-2 secretion, as well as suppression of both interferon γ (Th1) and IL-4 (Th2). Moreover, addition of anti–TGF-β partially reverses this T cell suppression. When CTLA-4 was cross-linked in T cell populations from TGF-β1 gene–deleted (TGF-β1^−/−) mice, the T cell responses were only suppressed 38% compared with 95% in wild-type mice. Our data demonstrate that engagement of CTLA-4 leads to CD4⁺ T cell production of TGF-β, which, in part, contributes to the downregulation of T cell activation. CTLA-4, through TGF-β, may serve as a counterbalance for CD28 costimulation of IL-2 and CD4⁺ T cell activation.     

Cytomegalovirus mediates expansion of IL-15–responsive innate-memory cells with SIV killing function

Interindividual immune variability is driven predominantly by environmental factors, including exposure to chronic infectious agents such as cytomegalovirus (CMV). We investigated the effects of rhesus CMV (RhCMV) on composition and function of the immune system in young macaques. Within months of infection, RhCMV was associated with impressive changes in antigen presenting cells, T cells, and NK cells—and marked expansion of innate-memory CD8⁺ T cells. These cells express high levels of NKG2A/C and the IL-2 and IL-15 receptor beta chain, CD122. IL-15 was sufficient to drive differentiation of the cells in vitro and in vivo. Expanded NKG2A/C⁺CD122⁺CD8⁺ T cells in RhCMV-infected macaques, but not their NKG2-negative counterparts, were endowed with cytotoxicity against class I–deficient K562 targets and prompt IFN-γ production in response to stimulation with IL-12 and IL-18. Because RhCMV clone 68-1 forms the viral backbone of RhCMV-vectored SIV vaccines, we also investigated immune changes following administration of RhCMV 68-1–vectored SIV vaccines. These vaccines led to impressive expansion of NKG2A/C⁺CD8⁺ T cells with capacity to inhibit SIV replication ex vivo. Thus, CMV infection and CMV-vectored vaccination drive expansion of functional innate-like CD8 cells via host IL-15 production, suggesting that innate-memory expansion could be achieved by other vaccine platforms expressing IL-15.     

ESAT-6–dependent cytosolic pattern recognition drives noncognate tuberculosis control in vivo

IFN-γ is a critical mediator of host defense against Mycobacterium tuberculosis (Mtb) infection. Antigen-specific CD4⁺ T cells have long been regarded as the main producer of IFN-γ in tuberculosis (TB), and CD4⁺ T cell immunity is the main target of current TB vaccine candidates. However, given the recent failures of such a TB vaccine candidate in clinical trials, strategies to harness CD4-independent mechanisms of protection should be included in future vaccine design. Here, we have reported that noncognate IFN-γ production by Mtb antigen–independent memory CD8⁺ T cells and NK cells is protective during Mtb infection and evaluated the mechanistic regulation of IFN-γ production by these cells in vivo. Transfer of arenavirus- or protein-specific CD8⁺ T cells or NK cells reduced the mortality and morbidity rates of mice highly susceptible to TB in an IFN-γ–dependent manner. Secretion of IFN-γ by these cell populations required IL-18, sensing of mycobacterial viability, Mtb protein 6-kDa early secretory antigenic target–mediated (ESAT-6–mediated) cytosolic contact, and activation of NLR family pyrin domain–containing protein 3 (NLRP3) inflammasomes in CD11c⁺ cell subsets. Neutralization of IL-18 abrogated protection in susceptible recipient mice that had received noncognate cells. Moreover, improved Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccine–induced protection was lost in the absence of ESAT-6–dependent cytosolic contact. Our findings provide a comprehensive mechanistic framework for antigen-independent IFN-γ secretion in response to Mtb with critical implications for future intervention strategies against TB.     

Interleukin 12 (IL-12) Is Crucial to the Development of Protective Immunity in Mice Intravenously Infected with Mycobacterium tuberculosis

Immunity to Mycobacterium tuberculosis infection is associated with the emergence of protective CD4 T cells that secrete cytokines, resulting in activation of macrophages and the recruitment of monocytes to initiate granuloma formation. The cytokine-mediating macrophage activation is interferon-γ (IFN-γ), which is largely dependent on interleukin-12 (IL-12) for its induction. To address the role of IL-12 in immunity to tuberculosis, IL-12 p40^−/− mice were infected with M. tuberculosis and their capacity to control bacterial growth and other characteristics of their immune response were determined. The IL-12 p40^−/− mice were unable to control bacterial growth and this appeared to be linked to the absence of both innate and acquired sources of IFN-γ. T cell activation as measured by delayed type hypersensitivity and lymphocyte accumulation at the site of infection were both markedly reduced in the IL-12 p40^−/− mice. Therefore, IL-12 is essential to the generation of a protective immune response to M. tuberculosis, with its main functions being the induction of the expression of IFN-γ and the activation of antigen-specific lymphocytes capable of creating a protective granuloma.     

Tpl2 kinase regulates T cell interferon-γ production and host resistance to Toxoplasma gondii

Tpl2 (Tumor progression locus 2), also known as Cot/MAP3K8, is a hematopoietically expressed serine-threonine kinase. Tpl2 is known to have critical functions in innate immunity in regulating tumor necrosis factor–α, Toll-like receptor, and G protein–coupled receptor signaling; however, our understanding of its physiological role in T cells is limited. We investigated the potential roles of Tpl2 in T cells and found that it was induced by interleukin-12 in human and mouse T cells in a Stat4-dependent manner. Deficiency of Tpl2 was associated with impaired interferon (IFN)-γ production. Accordingly, Tpl2^−/− mice had impaired host defense against Toxoplasma gondii with reduced parasite clearance and decreased IFN-γ production. Furthermore, reconstitution of Rag2^−/− mice with Tpl2-deficient T cells followed by T. gondii infection recapitulated the IFN-γ defect seen in the Tpl2-deficient mice, confirming a T cell–intrinsic defect. CD4⁺ T cells isolated from Tpl2^−/− mice showed poor induction of T-bet and failure to up-regulate Stat4 protein, which is associated with impaired TCR-dependent extracellular signal-regulated kinase activation. These data underscore the role of Tpl2 as a regulator of T helper cell lineage decisions and demonstrate that Tpl2 has an important functional role in the regulation of Th1 responses.     

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.     

Virus-specific CD8+ T Lymphocytes Downregulate T Helper Cell Type 2 Cytokine Secretion and Pulmonary Eosinophilia during Experimental Murine Respiratory Syncytial Virus Infection

T lymphocytes play a pivotal role in the immune response during viral infections. In a murine model of experimental respiratory syncytial virus (RSV) infection, mice sensitized to either of the two major glycoproteins of RSV develop distinct patterns of cytokine secretion and lung inflammation upon subsequent RSV infection. Mice sensitized to RSV-G (attachment) glycoprotein exhibit a strong interleukin (IL)-4 and IL-5 response and develop pulmonary eosinophilia, whereas mice sensitized to RSV-F (fusion) glycoprotein develop a predominantly T helper cell (Th)1 response and pulmonary inflammation characterized by mononuclear cell infiltration. In this study, we examined the potential role of virus-specific CD8⁺ T cytolytic T cells on the differentiation and activation of functionally distinct CD4⁺ T cells specific to these viral glycoproteins. Mice primed with recombinant vaccinia virus expressing RSV-F glycoprotein mounted a strong RSV-specific, MHC class I–restricted cytolytic response, whereas priming with recombinant vaccinia virus expressing RSV-G glycoprotein failed to elicit any detectable cytolytic response. Priming for a RSV-specific CD8⁺ T cell response, either with a recombinant vaccinia virus expressing RSV-G glycoprotein in which a strong CD8⁺ T cell epitope from RSV-M2 (matrix) protein has been inserted or with a combination of vaccinia virus expressing the matrix protein and the RSV-G glycoprotein, suppressed the eosinophil recruitment into the lungs of these mice upon subsequent challenge with RSV. This reduction in pulmonary eosinophilia correlated with the suppression of Th2 type cytokine production. The importance of CD8⁺ T cells in this process was further supported by the results in CD8⁺ T cell deficient, β₂ microglobulin KO mice. In these mice, priming to RSV-F glycoprotein (which in normal mice primed for a strong cytolytic response and a pulmonary infiltrate consisting primarily of mononuclear cells on RSV challenge) resulted in the development of marked pulmonary eosinophilia that was not seen in mice with an intact CD8⁺ T cell compartment. These results indicate that CD8⁺ T cells may play an important role in the regulation of the differentiation and activation of Th2 CD4⁺ T cells as well as the recruitment of eosinophils into the lungs during RSV infection.Multiple arms of the immune system are activated in response to infection by foreign organisms. The outcomes of the infection, such as the clearance of the organisms and the generation of tissue injury, depend on these immune effector mechanisms that are mobilized against the pathogen. The role of T cells in the immune response to viral infections has been clearly established (1, 2). Mature T cells that express α/β T cell receptors can be divided into cells expressing either CD4 or CD8 surface antigen. CD8⁺ T cells recognize processed antigen in the context of MHC class I molecules, whereas CD4⁺ T cells recognize peptides in the context of MHC class II molecules (3, 4). The conventional view of CD8⁺ T cells has been primarily the killing of virally infected cells by direct cytolysis or by cytokines secreted by these T cells such as IFN-γ and TNF (1, 2). Functional subsets of CD4⁺ T cells have been described based on the cytokines produced by these cells, Th1 CD4⁺ T cells that secrete IL-2 and IFN-γ and Th2 T cells that secrete IL-4 and IL-5 (5, 6). The physiological relevance of these subsets of T cells with diverse functions have been shown in several models of viral infection including respiratory syncytial virus (RSV)¹.In the murine model of RSV infection, the roles of T cells and their products in the eradication of the virus as well as the pathogenesis of lung inflammation have been well documented (7–12). Recent studies have shown that mice sensitized to either of the two major glycoproteins of this virus developed distinct lung pathology when subsequently infected with RSV (12–15). Mice sensitized to the attachment glycoprotein (G) of RSV developed pulmonary eosinophilia that correlated with a strong induction of Th2 type response, whereas mice primed to the fusion (F) glycoprotein mounted a weak Th2 response and developed lung inflammation characterized by mononuclear cell infiltration. The underlying mechanisms that lead to these apparent distinct patterns of cytokine production and lung injury still remain unclear.The process by which CD4⁺ T cells mature and differentiate has been the subject of intense investigation (16). CD4⁺ T cells can be induced to differentiate into effector cells of either the Th1 or Th2 subsets, depending on the milieu in which these cells are activated. Cytokines such as IL-4 and IL-12 have been shown to play a crucial part in the regulation of CD4⁺ T cell differentiation (17–19). The presence of certain immune effector functions during the differentiation and expansion of CD4⁺ T cells may potentially be a key factor in determining the functional phenotypes acquired by these cells. In the murine model of experimental RSV infection, one important difference in the immune response to F and G glycoprotein of RSV is the lack of MHC class I–restricted cytolytic response to the G glycoprotein (13, 20). In the following studies, we began to investigate the impact of CD8⁺ T cells and MHC class I–restricted CTL responses on cytokine secretion and the subsequent development of pulmonary eosinophilia during experimental murine RSV infection. We have found that the induction of CD8⁺ T cell response to RSV proteins resulted in dramatically decreased levels of Th2 type cytokines and eosinophil recruitment into the lungs of RSV-infected mice previously sensitized to the RSV-G glycoprotein. The possible mechanisms underlying these observations and their potential significance are discussed.     

Endogenous Interleukin 4 Is Required for Development of Protective CD4+ T Helper Type 1 Cell Responses to Candida albicans

Interleukin (IL)-4–deficient mice were used to assess susceptibility to systemic or gastrointestinal Candida albicans infections, as well as parameters of innate and elicited T helper immunity. In the early stage of systemic infection with virulent C. albicans, an unopposed interferon (IFN)-γ response renders IL-4–deficient mice more resistant than wild-type mice to infection. Yet, IL-4–deficient mice failed to efficiently control infection in the late stage and succumbed to it. Defective IFN-γ and IL-12 production, but not IL-12 responsiveness, was observed in IL-4–deficient mice that failed to mount protective T helper type 1 cell (Th1)-mediated acquired immunity in response to a live vaccine strain of the yeast or upon mucosal immunization in vivo. In vitro, IL-4 primed neutrophils for cytokine release, including IL-12. However, late treatment with exogenous IL-4, while improving the outcome of infection, potentiated CD4⁺ Th1 responses even in the absence of neutrophils. These findings indicate that endogenous IL-4 is required for the induction of CD4⁺ Th1 protective antifungal responses, possibly through the combined activity on cells of the innate and adaptive immune systems.