Abstract The TCR signals for the release of CD4 effector function are poorly understood. Itk plays an essential role in Th2, but not Th1, responses. However, when Itk is required during Th2 development is unclear. We followed the fate of Itk-deficient T cells during Th2 development in vitro and in vivo using an IL-4/GFP reporter. Surprisingly, a similar frequency of itk−/− CD4+ cells differentiated and committed to the Th2 lineage as wild-type cells. However, Itk-deficient Th2 cells failed to exert effector function upon TCR triggering. Loss of function was marked by defective transcriptional enhancement of Th2 cytokines and GATA3. IL-4 production in itk−/− Th2s could be rescued by the expression of kinase-active Itk. Thus, Itk is necessary for the release, but not gain, of Th2 function. We suggest that the liberation of effector function is tightly controlled through qualitative changes in TCR signals, facilitating postdifferentiation regulation of cytokine responses.
Compartmentalization of the immune response ensures
tight regulation of T cell activation in the lymph node (LN) and
precise effector T cell delivery and function at sites of
inflammation. During differentiation within the LN, functionally
discrete effector T cells acquire distinct homing properties that
direct them to sites of inflammation. We show that such
tissue-specific accumulation of cytokine secreting effector T cells
can be subverted by a pathogen at the infected tissue site. On
Leishmania major (L. major) infection, the draining LN (dLN) of
both susceptible and resistant mice contained both IL-4 and
IFNγ-producing T cells early in infection. In contrast, primarily
IL-4 producing cells were found in the infected tissue. Despite a
striking absence of IFNγ producers in the L. major-infected tissue,
L. major-specific IFNγ effectors were readily detectable at an
independent inflammatory site. Recruitment of non-Leishmania
specific effectors to the infection site was similarly restricted.
Importantly, provision of strong inflammatory signals (CpG) failed
to disrupt the selective cytokine production at the L. major
infection site, suggesting that L. major actively modifies the
local milieu. To formally test this idea, we assessed the ability
of the L. major infected site to recruit a third-party effector
population. Mice were infected with L. major in one ear and
immunized with ovalbumin (OVA) in the contralateral ear. Two weeks
after infection, mice were rechallenged with soluble OVA directly
in the L. major infected ear. Indeed, while both anti-OVA IL-4 and
IFNγ producers were found in the OVA-immunized ear and dLN, only
IL-4 producing, OVA specific cells were found in the L.
major-infected tissue. Mechanistically, IFNγ producers could fail
to be recruited, be recruited but not be retained or be recruited
and then functionally modified or terminated. Our data suggests a
role for L. major in shaping the early chemokine microenvironment.
Early upon infection, we observed a striking restriction in
chemokine expression at the infection site; expression of CCL7 but
not Th1-attracting chemokines. Moreover, L. major potently
inhibited CpG-induction of various Th1-attracting chemokines in
part through a direct action of the parasite on the infected
macrophage. These data implicate a role for the local chemokine
milieu in shaping the resulting cytokine repertoire at the site of
infection. In addition we have developed techniques that will
enable us to track effector cell function and localization within
the whole mouse. The use of fluorescent parasites in combination
with cytokine reporter mice, whole mount immunohistochemistry and
bioluminescence imaging will allow us to monitor the
recruitment/retention and functional status of antigen specific
effector cells at the site of infection. We suggest that the
pathogen-driven restriction to non-pathogenic IL-4 producing cells
enables L. major to gain early host residency. This work
demonstrates the need to understand the immune response at infected
tissue sites in addition…
Abstract T cell–APC interactions are essential for the initiation of effector responses against foreign and self-antigens, but the role of these interactions in generating different populations of effector T cells in vivo remains unclear. Using a model of CD4+ T cell responses to a systemic self-antigen without adjuvants or infection, we demonstrate that activation of APCs augments Th17 responses much more than Th1 responses. Recognition of systemic Ag induces tolerance in self-reactive CD4+ T cells, but induction of CD40 signaling, even under tolerogenic conditions, results in a strong, Ag-specific IL-17 response without large numbers of IFN-γ–producing cells. Transfer of the same CD4+ T cells into lymphopenic recipients expressing the self-antigen results in uncontrolled production of IL-17, IFN-γ, and systemic inflammation. If the Ag-specific T cells lack CD40L, production of IL-17 but not IFN-γ is decreased, and the survival time of recipient mice is significantly increased. In addition, transient blockade of the initial MHC class II-dependent T cell–APC interaction results in a greater reduction of IL-17 than of IFN-γ production. These data suggest that Th17 differentiation is more sensitive to T cell interactions with APCs than is the Th1 response, and interrupting this interaction, specifically the CD40 pathway, may be key to controlling Th17-mediated autoimmunity.
Abstract BALB/c IL-2-KO mice develop systemic autoimmunity, dying within 4 wks from autoimmune hemolytic anemia. Disease in these mice is Th1-mediated, and IFNγ production is required for early autoimmunity. We asked whether APC are required for IFNγ production by T cells in the IL-2-KO mouse. Our data show that APC:T cell interactions through B7, but not via B cells, are necessary for IFNγ production in the absence of IL-2, suggesting that dendritic cells (DC) mediate this alteration. Disease is marked by DC accumulation, activation and elevated cytokine production, including IL-12 and type1-interferons. The depletion of either conventional (cDC) or plasmacytoid (pDC) significantly augmented the survival of IL-2-KO mice, demonstrating that DC contribute to the progression of autoimmunity. In the absence of IL-12, T cell activation and disease is only moderately altered, thus Th1 differentiation and IFNγ induction as a part of the underlying kinetics of autoimmunity is primarily IL-12-independent. Elimination of type 1 interferon signals in addition to IL-12 further augmented survival, indicating that cytokines derived from both pDC and cDC contribute to disease severity. Enhanced DC function is not dependent on T cell activation as DC in an environment without overt T cell activation are still functionally activated. Overall, our data suggest that DC activation can be an initiating event during the development of disease and thus DC are critical regulators of autoimmune development.
Interleukin 4 (IL-4) plays a central role in the orchestration of Type 2 immunity. During T cell activation in the lymph node, IL-4 promotes Th2 differentiation and inhibits Th1 generation. In the inflamed tissue, IL-4 signals promote innate and adaptive Type-2 immune recruitment and effector function, positively amplifying the local Th2 response. In this study, we identify an additional negative regulatory role for IL-4 in limiting the recruitment of Th1 cells to inflamed tissues. To test IL-4 effects on inflammation subsequent to Th2 differentiation, we transiently blocked IL-4 during ongoing dermal inflammation (using anti-IL-4 mAb) and analyzed changes in gene expression. Neutralization of IL-4 led to the upregulation of a number of genes linked to Th1 trafficking, including CXCR3 chemokines, CCL5 and CCR5 and an associated increase in IFNγ, Tbet and TNFα genes. These gene expression changes correlated with increased numbers of IFNγ-producing CD4+ T cells in the inflamed dermis. Moreover, using an adoptive transfer approach to directly test the role of IL-4 in T cell trafficking to the inflamed tissues, we found IL-4 neutralization led to an early increase in Th1 cell recruitment to the inflamed dermis. These data support a model whereby IL-4 dampens Th1-chemokines at the site of inflammation limiting Th1 recruitment. To determine biological significance, we infected mice with Leishmania major, as pathogen clearance is highly dependent on IFNγ-producing CD4+ T cells at the infection site. Short-term IL-4 blockade in established L. major infection led to a significant increase in the number of IFNγ-producing CD4+ T cells in the infected ear dermis, with no change in the draining LN. Increased lymphocyte influx into the infected tissue correlated with a significant decrease in parasite number. Thus, independent of IL-4's role in the generation of immune effectors, IL-4 attenuates lymphocyte recruitment to the inflamed/infected dermis and limits pathogen clearance.
Abstract The initial encounter of a T cell with its antigen-presenting cell (APC) defines a major regulation point, relaying information to the responding T cell. We have assessed the interaction of T cells with APCs under conditions of tolerance and activation, by evaluating the response of auto-reactive CD4+ T cells upon recognition of self-antigen. In this model DO11.10 CD4+ T cells specific for OVA peptide (323-339) are transferred into transgenic mice, which express OVA as a systemically secreted soluble protein (sOVA-Tg). Previous studies in the lab have shown that in a "full" sOVA host, the auto-reactive T cells become tolerant, while in an "empty" or lymphopenic sOVA Rag-/- host, T cells respond with uncontrolled activation and host mice develop autoimmunity. Using 2-photon microscopy we have evaluated the velocity and movement during the initial encounter of T cells with APCs under conitions of tolerance or activation. Our data suggests that under both conditions the antigen specific cells initially stop on APCs, this interaction is retained in sOVARag-/- (autoimmune) mice but lost in sOVA (tolerant) hosts. In addition, the initial activation through the T cell receptor, as determined by pS6 staining, is comparable during early activation. This signal is retained in sOVARag-/- hosts but in contrast rapidly declines in sOVA hosts. These data show that the loss of early activation may define the tolerant phenotype. Understanding the interactions of self-reactive T cells with APCs presenting self-antigen is key to understanding the induction of tolerance vs. immunity and how this balance is maintained is integral for the design of therapies in the future. Research Support-NIH (F32)
In helper T cells, IL-13 is traditionally considered a Th2-type cytokine that is coexpressed with IL-4. Using mouse models of immunization and autoimmunity, we demonstrate that IL-13 is frequently uncoupled from IL-4, and that it can be produced by both IFN-γ(+) Th1 cells and IL-17(+) Th17 cells. We report that these IL-13-producing Th1 and Th17 cells are distinct from classical IL-4(+) Th2 cells and that they are relatively common, appearing in the context of both protective and pathogenic T-cell responses. We also demonstrate that IL-13 and Th2-type cytokines can have important consequences in Th1- and Th17-dominated settings, such as lymphopenia-induced autoimmune disease, where they can be either pro- or anti-inflammatory, depending on whether they act on innate or adaptive immune cells. Taken together, our studies indicate that IL-13 production is more widespread than previously appreciated and that blocking this cytokine may have therapeutic benefits even in settings where traditional IL-4-driven Th2-type responses are not evident.
The early events that determine the decision between lymphocyte tolerance and activation are not well-understood. Using a model of systemic self-antigen recognition by CD4 + T cells, we show, using single-cell biochemical analyses, that tolerance is characterized by transient signaling events downstream of T-cell receptor engagement in the mammalian target of rapamycin (mTOR) and NF-κB pathways. Parallel studies done by live cell imaging show that the key difference between tolerance and activation is the duration of the T cell–antigen presenting cell (APC) interaction, as revealed by stable T-cell immobilization on antigen encounter. Brief T cell–APC interactions result in tolerance, and prolonged interactions are associated with activation and the development of effector cells. These studies show that the duration of T cell–APC interactions and magnitude of associated TCR-mediated signaling are key determinants of lymphocyte tolerance vs. activation.
