Autocrine IFN-γ promotes naive CD8 T cell differentiation and synergizes with IFN-α to stimulate strong function

Autocrine IFN-γ signaling is important for CD4 differentiation to Th1 effector cells, but it has been unclear whether it contributes to CD8 T cell differentiation. We show in this paper that naive murine CD8 T cells rapidly and transiently produce low levels of IFN-γ upon stimulation with Ag and B7-1, with production peaking at ～8 h and declining by 24 h. The autocrine IFN-γ signals for upregulation of expression of T-bet and granzyme B and induces weak cytolytic activity and effector IFN-γ production. IFN-α acts synergistically with IFN-γ to support development of strong effector functions, whereas IL-12 induces high T-bet expression and strong function in the absence of IFN-γ signaling. Thus, IFN-γ is not only an important CD8 T cell effector cytokine, it is an autocrine/paracrine factor whose contributions to differentiation vary depending on whether the response is supported by IL-12 or type I IFN.     

High TCR stimuli prevent induced regulatory T cell differentiation in a NF-κB-dependent manner

The concentration of Ag or mitogenic stimuli is known to play an important role in controlling the differentiation of naive CD4(+) T cells into different effector phenotypes. In particular, whereas TCR engagement at low Ag doses in the presence of TGF-β and IL-2 can promote differentiation of Foxp3-expressing induced regulatory T cells (iTregs), high levels of Ag have been shown in vitro and in vivo to prevent Foxp3 upregulation. This tight control of iTreg differentiation dictated by Ag dose most likely determines the quality and duration of an immune response. However, the molecular mechanism by which this high-dose inhibition of Foxp3 induction occurs is not well understood. In this study, we demonstrate that when cells are in the presence of CD28 costimulation, TCR-dependent NF-κB signaling is essential for Foxp3 inhibition at high doses of TCR engagement in mouse T cells. Prevention of Foxp3 induction depends on the production of NF-κB-dependent cytokines by the T cells themselves. Moreover, T cells that fail to upregulate Foxp3 under iTreg-differentiating conditions and high TCR stimulation acquire the capacity to make TNF and IFN-γ, as well as IL-17 and IL-9. Thus, NF-κB helps T cells control their differentiation fate in a cell-intrinsic manner and prevents peripheral iTreg development under conditions of high Ag load that may require more vigorous effector T cell responses.     

Outer membrane protein A (OmpA) of Shigella flexneri 2a links innate and adaptive immunity in a TLR2-dependent manner and involvement of IL-12 and nitric oxide

We determine that OmpA of Shigella flexneri 2a is recognized by TLR2 and consequently mediates the release of proinflammatory cytokines and activates NF-κB in HEK 293 cells transfected with TLR2. We also observe that in RAW macrophages TLR2 is essential to instigate the early immune response to OmpA via NF-κB activation and secretion of cytokines and NO. Consistent with these results, TLR2 knockdown using siRNA abolishes the initiation of immune responses. Processing and presentation of OmpA depend on TLR2; MHCII presentation of the processed antigen and expression of CD80 significantly attenuated in TLR2 knockdown macrophages. The optimum production of IFN-γ by the macrophages:CD4(+) T cells co-culture depends on both TLR2 activation and antigen presentation. So, TLR2 is clearly recognized as a decisive factor in initiating host innate immune response to OmpA for the development of CD4(+) T cell adaptive response. Furthermore, we demonstrate in vivo that intranasal immunization of mice with OmpA selectively enhances the release of IFN-γ and IL-2 by CD4(+) T cells. Importantly, OmpA increases the level of IFN-γ production in Ag-primed splenocytes. The addition of neutralizing anti-IL-12p70 mAb to cell cultures results in the decreased release of OmpA-enhanced IFN-γ by Ag-primed splenocytes. Moreover, coincubation with OmpA-pretreated macrophages enhances the production of IFN-γ by OmpA-primed CD4(+) T cells, representing that OmpA may enhance IFN-γ expression in CD4(+) T cells through the induction of IL-12 production in macrophages. These results demonstrate that S. flexneri 2a OmpA may play a critical role in the development of Th1 skewed adaptive immune response.     

Amelioration of experimental autoimmune encephalomyelitis by plumbagin through down-regulation of JAK-STAT and NF-κB signaling pathways

Plumbagin (PL), a herbal compound derived from roots of the medicinal plant Plumbago zeylanica, has been shown to have immunosuppressive properties. Present report describes that PL is a potent novel agent in control of encephalitogenic T cell responses and amelioration of mouse experimental autoimmune encephalomyelitis (EAE), through down-regulation of JAK-STAT pathway. PL was found to selectively inhibit IFN-γ and IL-17 production by CD4(+) T cells, which was mediated through abrogated phosphorylation of JAK1 and JAK2. Consistent with IFN-γ and IL-17 reduction was suppressed STAT1/STAT4/T-bet pathway which is critical for Th1 differentiation, as well as STAT3/ROR pathway which is essential for Th17 differentiation. In addition, PL suppressed pro-inflammatory molecules such as iNOS, IFN-γ and IL-6, accompanied by inhibition of IκB degradation as well as NF-κB phosphorylation. These data give new insight into the novel immune regulatory mechanism of PL and highlight the great value of this kind of herb compounds in probing the complex cytokine signaling network and novel therapeutic targets for autoimmune diseases.     

Functional CD8 T cell memory responding to persistent latent infection is maintained for life

Aging is associated with depressed naive T cell responses, but it is less clear whether T cell memory established early in life also becomes impaired with age. This is particularly important for T cells responding to latent persistent infection, which need to remain functional and capable of controlling the infection over the lifetime; however, repeated stimulation over the lifetime may dysregulate their maintenance or function, potentially contributing to impaired immunity in the elderly. Systemic infection with HSV-1, a persistent latent virus, is associated with memory inflation of virus-specific CD8 T cells. We tested how these inflated memory cells are maintained from adulthood into old age. We found no significant differences in the numbers (i.e., blood, spleen), ex vivo Ag-specific IFN-γ production, and in vivo recall response to HSV-1 (i.e., proliferation, IFN-γ production, cytolysis) between adult and old memory T cells. There was a discrete shift from dominantly effector memory phenotype in the adults to a central memory-like phenotype in the old mice, with fewer old cells expressing the killer cell lectin-like receptor G1 (KLRG1). Adult and old KLRG1(+) memory CD8 T cells were functionally identical: both produced IFN-γ but could minimally proliferate in response to viral challenge. Interestingly, regardless of age, KLRG1(+) cells retained the ability to proliferate and survive in response to homeostatic signals, both in vitro (culture with IL-7 and IL-15) and in vivo (expansion following transfer into lymphopenic recipients). This finding demonstrates that functional effector memory T cells, including those expressing KLRG-1, are maintained and are functional for life, despite the presence of persistent viral infection.     

Extracellular purine metabolism and signaling of CD73-derived adenosine in murine Treg and Teff cells

CD73-derived adenosine acts as potent inhibitor of inflammation, and regulatory T cells (Treg) have been shown to express CD73 as a novel marker. This study explored the role of endogenously formed adenosine in modulating NF-κB activity and cytokine/chemokine release from murine Treg and effector T cells (Teff) including key enzymes/purinergic receptors of extracellular ATP catabolism. Stimulating murine splenocytes and CD4(+) T cells with anti-CD3/anti-CD28 significantly upregulated activated NF-κB in CD73(-/-) T cells (wild type: 4.36 ± 0.21; CD73(-/-): 6.58 ± 0.75; n = 4; P = 0.029). This was associated with an augmented release of proinflammatory cytokines IL-2, TNF-α, and IFN-γ. Similar changes were observed with the CD73 inhibitor APCP (50 μM) on NF-κB and IFN-γ in wild-type CD4(+) T-cells. Treatment of stimulated CD4(+) T-cells with adenosine (25 μM) potently reduced IFN-γ release which is mediated by adenosine A2a receptors (A2aR). AMP (50 μM) also reduced cytokine release which was not inhibited by APCP. In Teff, A2aR activation (CGS21680) potently inhibited the release of IL-1, IL-2, IL-3, IL-4, IL-12, IL-13, IFN-γ, TNF-α, granulocyte-macrophage colony-stimulating factor (GM-CSF), CCL3, and CCL4. However, in Treg, CGS21680 did not alter cytokine/chemokine release. In summary, CD73-derived adenosine tonically inhibits active NF-κB in CD4(+) T-cells, thereby modulating the release of a broad spectrum of proinflammatory cytokines and chemokines. Downregulation of P2X7 and upregulation of CD73 in Treg after antigenic stimulation may be an important mechanism to maintain the ability of Treg to generate immunosuppressive adenosine.     

A biochemical signature for rapid recall of memory CD4 T cells

Mechanisms for the rapid recall response mediated by memory T cells remain unknown. In this study, we present a novel, multiparameter analysis of TCR-coupled signaling and function in resting and activated naive and memory CD4 T cells, revealing a biochemical basis for immunological recall. We identify a striking elevation in expression of the proximal tyrosine kinase Zap70 in resting Ag-specific and polyclonal mouse memory vs naive CD4 T cells that is stably maintained independent of protein synthesis. Elevated Zap70 protein levels control effector function as IFN-gamma production occurs exclusively from the Zap70(high) fraction of activated T cells in vitro and in vivo, and specific down-modulation of Zap70 expression in memory CD4 T cells by small interfering RNA or protein inhibition significantly reduces rapid IFN-gamma production. Downstream of Zap70, we show quantitative differences in distal phosphorylation associated with effector function in naive and memory subsets, with low accumulation of phosphorylation in memory T cells producing IFN-gamma at early time points, contrasting extensive phosphorylation associated with IFN-gamma production following sustained activation of naive T cells. Our results reveal a novel biochemical signature imparted to memory CD4 T cells enabling efficacious responses through increased Zap70 expression and reduced accumulation of downstream signaling events.     

CD4+ T Cell-Dependent IFN-γ Production by CD8+ Effector T Cells in Mycobacterium tuberculosis Infection

Both CD4(+) and CD8(+) T cells contribute to immunity to tuberculosis, and both can produce the essential effector cytokine IFN-γ. However, the precise role and relative contribution of each cell type to in vivo IFN-γ production are incompletely understood. To identify and quantitate the cells that produce IFN-γ at the site of Mycobacterium tuberculosis infection in mice, we used direct intracellular cytokine staining ex vivo without restimulation. We found that CD4(+) and CD8(+) cells were predominantly responsible for production of this cytokine in vivo, and we observed a remarkable linear correlation between the fraction of CD4(+) cells and the fraction of CD8(+) cells producing IFN-γ in the lungs. In the absence of CD4(+) cells, a reduced fraction of CD8(+) cells was actively producing IFN-γ in vivo, suggesting that CD4(+) effector cells are continually required for optimal IFN-γ production by CD8(+) effector cells. Accordingly, when infected mice were treated i.v. with an MHC-II-restricted M. tuberculosis epitope peptide to stimulate CD4(+) cells in vivo, we observed rapid activation of both CD4(+) and CD8(+) cells in the lungs. Indirect activation of CD8(+) cells was dependent on the presence of CD4(+) cells but independent of IFN-γ responsiveness of the CD8(+) cells. These data provide evidence that CD4(+) cell deficiency impairs IFN-γ production by CD8(+) effector cells and that ongoing cross-talk between distinct effector T cell types in the lungs may contribute to a protective immune response against M. tuberculosis. Conversely, defects in these interactions may contribute to susceptibility to tuberculosis and other infections.     

Functional responses and costimulator dependence of memory CD4+ T cells

To examine the functional characteristics of memory CD4+ T cells, we used an adoptive transfer system to generate a stable population of Ag-specific memory cells in vivo and compared their responses to Ag with those of a similar population of Ag-specific naive cells. Memory cells localized to the spleen and lymph nodes of mice and exhibited extremely rapid recall responses to Ag in vivo, leaving the spleen within 3-5 days of Ag encounter. Unlike their naive counterparts, memory cells produced effector cytokines (IFN-gamma, IL-4, IL-5) within 12-24 h of Ag exposure and did not require multiple cycles of cell division to do so. Memory cells proliferated at lower Ag concentrations than did naive cells, were less dependent on costimulation by B7 molecules, and independent of costimulation by CD40. Furthermore, effector cytokine production by memory cells also occurred in the absence of either B7 or CD40 costimulation. Lastly, memory cells were resistant to tolerance induction. Together, these findings suggest that the threshold for activation of memory CD4+ cells is lower than that of naive cells. This would permit memory cells to rapidly express their effector functions in vivo earlier in the course of a secondary immune response, when the levels of Ag and the availability of costimulation may be relatively low.     

OX40/OX40L costimulation affects induction of Foxp3+ regulatory T cells in part by expanding memory T cells in vivo

OX40 is a member of the TNFR superfamily and has potent T cell costimulatory activities. OX40 also inhibits the induction of Foxp3(+) regulatory T cells (Tregs) from T effector cells, but the precise mechanism of such inhibition remains unknown. In the present study, we found that CD4(+) T effector cells from OX40 ligand-transgenic (OX40Ltg) mice are highly resistant to TGF-beta mediated induction of Foxp3(+) Tregs, whereas wild-type B6 and OX40 knockout CD4(+) T effector cells can be readily converted to Foxp3(+) T cells. We also found that CD4(+) T effector cells from OX40Ltg mice are heterogeneous and contain a large population of CD44(high)CD62L(-) memory T cells. Analysis of purified OX40Ltg naive and memory CD4(+) T effector cells showed that memory CD4(+) T cells not only resist the induction of Foxp3(+) T cells but also actively suppress the conversion of naive CD4(+) T effector cells to Foxp3(+) Tregs. This suppression is mediated by the production of IFN-gamma by memory T cells but not by cell-cell contact and also involves the induction of T-bet. Importantly, memory CD4(+) T cells have a broad impact on the induction of Foxp3(+) Tregs regardless of their origins and Ag specificities. Our data suggest that one of the mechanisms by which OX40 inhibits the induction of Foxp3(+) Tregs is by inducing memory T cells in vivo. This finding may have important clinical implications in tolerance induction to transplanted tissues.     

Gamma interferon and perforin control the strength, but not the hierarchy, of immunodominance of an antiviral CD8+ T cell response

The two major antiviral effector mechanisms of CD8(+) T cells are thought to be perforin (Prf)-mediated cell lysis and gamma interferon (IFN-γ)-mediated induction of an antiviral state. By affecting the expression of proteins involved in antigen presentation, IFN-γ is also thought to shape the magnitude and specificity of the CD8(+) T cell response. Here we studied the roles of Prf and IFN-γ in shaping the effector and memory CD8(+) T cell responses to vaccinia virus (VACV). IFN-γ deficiency resulted in increased numbers of anti-VACV effector and memory CD8(+) T cells, which were partly dependent on increased virus loads. On the other hand, Prf-deficient mice showed an increase in the number of VACV-specific CD8(+) T cells only in the memory phase. Treatment of the mice with the antiviral drug cidofovir reduced the numbers of effector and memory cells closer to wild-type levels in IFN-γ-deficient mice and reduced the numbers of memory CD8(+) T cells to wild-type levels in Prf-deficient mice. These data suggest that virus loads are the main reason for the increased strength of the CD8 response in IFN-γ- and Prf-deficient mice. Neither Prf deficiency nor IFN-γ deficiency had an effect on the immunodominance hierarchy of five K(b)-restricted CD8(+) T cell determinants either during acute infection or after recovery. Thus, our work shows that CD8(+) T cell immunodominance during VACV infection is not affected by the effects of IFN-γ on the antigen presentation machinery.