Inhibition of autoimmune T cell responses in the DA rat by bone marrow-derived NK cells in vitro: implications for autoimmunity.

Regulation of the immune response is critical to homeostasis. While innate immunity can influence the development of adaptive immune responses, its role in regulation is less well understood. Recently, NK cells have been implicated in the control of experimental autoimmune encephalomyelitis, an animal model for multiple sclerosis. In this report, we show that rat bone marrow-derived NK cells exhibited potent inhibitory effects on T cell proliferation to both Con A as well as the central nervous system Ag myelin basic protein. There was also a significant decrease in both IFN-gamma and IL-10 production in vitro, whereas levels of the beta-chemokine monocyte chemoattractant protein-1 were significantly elevated. Flow cytometry studies suggest that the NK cells may play an important role in regulating both normal and autoimmune T cell responses by exerting a direct effect on activated, autoantigen-specific T cells.     

Initial and innate responses to viral infections--pattern setting in immunity or disease.

Host responses to infectious challenges include initial events elicited directly by agent structures distinct from host determinants, activation of innate immune system components by the products of initial events, and the shaping of downstream adaptive immunity by these initial/innate responses. The picture emerging from viral infections is that viral structures interact with intracellular signaling pathways to induce expression of the type 1 interferons, IFN-alpha/beta. In addition to mediating direct antiviral effects, these cytokines play dominant roles in regulating innate and adaptive immune responses to infection. In particular, IFN-alpha/beta acts to inhibit interleukin-12 (IL-12) expression and IL-12 activation of innate natural killer (NK) cell IFN-alpha production, while inducing NK cell cytotoxicity and proliferation, and promoting adaptive T cell IFN-alpha responses. Although certain viral infections do elicit initial/innate IL-12 and NK-cell-produced IFN-alpha, endogenous IFN-alpha/beta also controls the magnitudes of these responses. Thus, the pathways activated, to dominantly regulate innate and adaptive immune responses during viral infections, are being defined.     

Inhibitory effect of LXR activation on cell proliferation and cell cycle progression through lipogenic activity

Liver X receptor (LXR), a sterol-activated nuclear hormone receptor, has been implicated in cholesterol and fatty acid homeostasis via regulation of reverse cholesterol transport and de novo fatty acid synthesis. LXR is also involved in immune responses, including anti-inflammatory action and T cell proliferation. In this study, we demonstrated that activated LXR suppresses cell cycle progression and proliferation in certain cell types. Stimulation of LXR with synthetic ligand T0901317 or GW3965 inhibited cell growth rate and arrested the cell cycle at the G1/S boundary in several cells, such as RWPE1, THP1, SNU16, LNCaP, and HepG2. However, LXR ligands did not exhibit antiproliferative activity in PC3, HEK293, or HeLa cells. Interestingly, activated LXR-mediated cell cycle arrest is closely correlated with the lipogenic gene expression and triacylglyceride accumulation. In accordance with these findings, suppression of FAS via small-interference RNA (siRNA) partially alleviated the antiproliferative effect of LXR activation in RWPE1 cells. Together, these data suggest that LXR activation with its ligands inhibits cell proliferation and induces G1/S arrest through elevated lipogenic activity, thus proposing a novel effect of activated LXR on cell cycle regulation.     

Bromelain modulates T cell and B cell immune responses in vitro and in vivo

The ability to modulate immune responses is a major aim of many vaccine and immunotherapeutic development programs. Bromelain, a mixture of cysteine proteases, modulates immunological responses and has been proposed to be of clinical use. However, the identity of the immune cells affected by bromelain and the specific cellular functions that are altered remain poorly understood. To address these shortcomings in our knowledge, we have used both in vitro and in vivo immunological assays to study the effects of bromelain. We found that bromelain enhanced T cell receptor (TCR) and anti-CD28-mediated T cell proliferation in splenocyte cultures by increasing the costimulatory activity of accessory cell populations. However, despite increased T cell proliferation, bromelain concomitantly decreased IL-2 production in splenocyte cultures. Additionally, bromelain did not affect TCR and CD28-induced proliferation of highly purified CD4+ T cells, but did inhibit IL-2 production by these cells. In vivo, bromelain enhanced T-cell-dependent, Ag-specific, B cell antibody responses. Again, bromelain induced a concomitant decrease in splenic IL-2 mRNA accumulation in immunized mice. Together, these data show that bromelain can simultaneously enhance and inhibit T cell responses in vitro and in vivo via a stimulatory action on accessory cells and a direct inhibitory action on T cells. This work provides important insights into the immunomodulatory activity of bromelain and has important implications for the use of exogenous cysteine proteases as vaccine adjuvants or immunomodulatory agents.     

Phosphatidylserine regulates the maturation of human dendritic cells

Phosphatidylserine (PS), which is exposed on the surface of apoptotic cells, has been implicated in immune regulation. However, the effects of PS on the maturation and function of dendritic cells (DCs), which play a central role in both immune activation and regulation, have not been described. Large unilamellar liposomes containing PS or phosphatidylcholine were used to model the plasma membrane phospholipid composition of apoptotic and live cells, respectively. PS liposomes inhibited the up-regulation of HLA-ABC, HLA-DR, CD80, CD86, CD40, and CD83, as well as the production of IL-12p70 by human DCs in response to LPS. PS did not affect DC viability directly but predisposed DCs to apoptosis in response to LPS. DCs exposed to PS had diminished capacity to stimulate allogeneic T cell proliferation and to activate IFN-gamma-producing CD4(+) T cells. Exogenous IL-12 restored IFN-gamma production by CD4(+) T cells. Furthermore, activated CTLs proliferated poorly to cognate Ag presented by DCs exposed to PS. Our findings suggest that PS exposure provides a sufficient signal to inhibit DC maturation and to modulate adaptive immune responses.     

Selectively impaired CD8+ but not CD4+ T cell cycle arrest during priming as a consequence of dendritic cell interaction with plasmodium-infected red cells

Individuals living in malaria-endemic areas show generally low T cell responses to malaria Ags. In this study, we show murine dendritic cell (DC) interaction with parasitized erythrocytes (pRBC) arrested their maturation, resulting in impaired ability to stimulate naive, but not recall T cell responses in vitro and in vivo. Moreover, within the naive T cell population, pRBC-treated DC were selectively deficient in priming CD8(+) but not CD4(+) T cells. Indeed, DC that had taken up pRBC were shown for the first time to efficiently prime CD4(+) T cell responses to a known protective merozoite Ag, MSP4/5. In contrast, impaired priming resulted in decreases in both proliferation and cytokine production by CD8(+) T cells. Deficient priming was observed to both a model and a Plasmodium berghei-specific CD8(+) T cell epitope. The mechanisms underlying the inability of parasite-treated DC to prime CD8(+) T cells were explored. pRBC treatment of DC from wild-type C57BL/6, but not from IL-10 knockout animals, suppressed DC-mediated T cell priming across a Transwell, suggesting active IL-10-dependent suppression. CD8(+) T cells were arrested at the G(0) stage of the cell cycle after two cell divisions post-Ag stimulation. The proliferation arrest was partially reversible by the addition of IL-2 or IL-7 to responder cultures. These results suggest that in malaria-endemic areas, priming of CD8(+) T cell responses may be more difficult to induce via vaccination than the priming of CD4(+) T cells. Moreover, pathogens may selectively target the CD8(+) T cell arm of protective immunity for immune evasion.     

Selective expansion and partial activation of human NK cells and NK receptor-positive T cells by IL-2 and IL-15.

IL-2 and IL-15 are lymphocyte growth factors produced by different cell types with overlapping functions in immune responses. Both cytokines costimulate lymphocyte proliferation and activation, while IL-15 additionally promotes the development and survival of NK cells, NKT cells, and intraepithelial lymphocytes. We have investigated the effects of IL-2 and IL-15 on proliferation, cytotoxicity, and cytokine secretion by human PBMC subpopulations in vitro. Both cytokines selectively induced the proliferation of NK cells and CD56(+) T cells, but not CD56(-) lymphocytes. All NK and CD56(+) T cell subpopulations tested (CD4(+), CD8(+), CD4(-)CD8(-), alphabetaTCR(+), gammadeltaTCR(+), CD16(+), CD161(+), CD158a(+), CD158b(+), KIR3DL1(+), and CD94(+)) expanded in response to both cytokines, whereas all CD56(-) cell subpopulations did not. Therefore, previously reported IL-15-induced gammadelta and CD8(+) T cell expansions reflect proliferations of NK and CD56(+) T cells that most frequently express these phenotypes. IL-15 also expanded CD8alpha(+)beta(-) and Valpha24Vbeta11 TCR(+) T cells. Both cytokines stimulated cytotoxicity by NK and CD56(+) T cells against K562 targets, but not the production of IFN-gamma, TNF-alpha, IL-2, or IL-4. However, they augmented cytokine production in response to phorbol ester stimulation or CD3 cross-linking by inducing the proliferation of NK cells and CD56(+) T cells that produce these cytokines at greater frequencies than other T cells. These results indicate that IL-2 and IL-15 act at different stages of the immune response by expanding and partially activating NK receptor-positive lymphocytes, but, on their own, do not influence the Th1/Th2 balance of adaptive immune responses.     

Regulatory NK cells suppress antigen-specific T cell responses

The immune system has a variety of regulatory/suppressive processes, which are decisive for the development of a healthy or an allergic immune response to allergens. NK1 and NK2 subsets have been demonstrated to display counterregulatory and provocative roles in immune responses, similar to Th1 and Th2 cells. T regulatory cells suppressing both Th1 and Th2 responses have been the focus of intensive research during the last decade. In this study, we aimed to investigate regulatory NK cells in humans, by characterization of NK cell subsets according to their IL-10 secretion property. Freshly purified IL-10-secreting NK cells expressed up to 40-fold increase in IL-10, but not in the FoxP3 and TGF-beta mRNAs. PHA and IL-2 stimulation as well as vitamin D3/dexamethasone and anti-CD2/CD16 mAbs are demonstrated to induce IL-10 expression in NK cells. The effect of IL-10+ NK cells on Ag-specific T cell proliferation has been examined in bee venom major allergen, phospholipase A2- and purified protein derivative of Mycobecterium bovis-induced T cell proliferation. IL-10+ NK cells significantly suppressed both allergen/Ag-induced T cell proliferation and secretion of IL-13 and IFN-gamma, particularly due to secreted IL-10 as demonstrated by blocking of the IL-10 receptor. These results demonstrate that a distinct small fraction of NK cells display regulatory functions in humans.     

Dendritic cells (DC) promote natural killer (NK) cell functions: dynamics of the human DC/NK cell cross talk

Dendritic cells (DC) were originally found critical in the setting of cognate immune responses. We first demonstrated that DC can also induce mouse NK cell activation and NK cell dependent-antitumor effects in mice. Here we analyzed the dynamics between DC and NK cells in human in vitro model systems. In the absence of LPS, DC do not trigger resting NK cells. Conversely, in the presence of LPS, resting bulk NK cells interacting with DC acquire CD25 and CD69 surface expression, produce high levels of IFN-gamma and lyse DAUDI cells. On activated IL-2 dependent NK cell lines, regardless of their differentiation stage, DC maintain or enhance NK cell proliferation and effector functions in the absence of exogenous cytokines. While IL-12, IL-15 and IL-18 are not critical, a direct cell-to-cell contact is mandatory for NK activation by DC and required for optimal proliferation. These data imply that DC also modulate human NK cell innate effector functions.     

Cutting Edge: Innate production of IFN-gamma by NK cells is independent of epigenetic modification of the IFN-gamma promoter

The ability of NK and T cells to produce IFN-gamma is critical for resistance to numerous intracellular pathogens but the kinetics of these responses differ. Consistent with this is a requirement for naive T cells to become activated and undergo proliferation-dependent epigenetic changes to the IFN-gamma locus that allow them to produce IFN-gamma. The data presented here reveal that unlike T cells, murine NK cells produce IFN-gamma under conditions of short-term cytokine stimulation, and these events are independent of proliferation and cell cycle progression. Furthermore, analysis of the IFN-gamma locus in NK cells reveals that this locus is constitutively demethylated. The finding that NK cells do not need to remodel the IFN-gamma locus to produce IFN-gamma, either because they do not exhibit epigenetic repression or they have undergone prior remodeling during development, provides a molecular basis for the innate and adaptive regulation of the production of this cytokine.     

Regulation of T cell priming by lymphoid stroma

The priming of immune T cells by their interaction with dendritic cells (DCs) in lymph nodes (LN), one of the early events in productive adaptive immune responses, occurs on a scaffold of lymphoid stromal cells, which have largely been seen as support cells or sources of chemokines and homeostatic growth factors. Here we show that murine fibroblastic reticular cells (FRCs), isolated from LN of B6 mice, play a more direct role in the immune response by sensing and modulating T cell activation through their upregulation of inducible nitric oxide synthase (iNOS) in response to early T cell IFNγ production. Stromal iNOS, which only functions in very close proximity, attenuates responses to inflammatory DC immunization but not to other priming regimens and preferentially affects Th1 cells rather than Th2. The resultant nitric oxide production does not affect T cell-DC coupling or initial calcium signaling, but restricts homotypic T cell clustering, cell cycle progression, and proliferation. Stromal feedback inhibition thus provides basal attenuation of T cell responses, particularly those characterized by strong local inflammatory cues.     

CD4+CD25+ T regulatory cells suppress NK cell-mediated immunotherapy of cancer

CD4+CD25+ regulatory T cells (Treg) that suppress T cell-mediated immune responses may also regulate other arms of an effective immune response. In particular, in this study we show that Treg directly inhibit NKG2D-mediated NK cell cytotoxicity in vitro and in vivo, effectively suppressing NK cell-mediated tumor rejection. In vitro, Treg were shown to inhibit NKG2D-mediated cytolysis largely by a TGF-beta-dependent mechanism and independently of IL-10. Adoptively transferred Treg suppressed NK cell antimetastatic function in RAG-1-deficient mice. Depletion of Treg before NK cell activation via NKG2D and the activating IL-12 cytokine, dramatically enhanced NK cell-mediated suppression of tumor growth and metastases. Our data illustrate at least one mechanism by which Treg can suppress NK cell antitumor activity and highlight the effectiveness of combining Treg inhibition with subsequent NK cell activation to promote strong innate antitumor immunity.     

Cross-talk between activated human NK cells and CD4+ T cells via OX40-OX40 ligand interactions

It is important to understand which molecules are relevant for linking innate and adaptive immune cells. In this study, we show that OX40 ligand is selectively induced on IL-2, IL-12, or IL-15-activated human NK cells following stimulation through NKG2D, the low affinity receptor for IgG (CD16) or killer cell Ig-like receptor 2DS2. CD16-activated NK cells costimulate TCR-induced proliferation, and IFN-gamma produced by autologous CD4+ T cells and this process is dependent upon expression of OX40 ligand and B7 by the activated NK cells. These findings suggest a novel and unexpected link between the natural and specific immune responses, providing direct evidence for cross-talk between human CD4+ T cells and NK receptor-activated NK cells.     

Regulation of T cell activation by TLR ligands

Regulatory T cells (Treg) maintain peripheral tolerance and play a critical role in the control of the immune response in infection, tumor defense, organ transplantation and allergy. CD4(+)CD25(high) Treg suppress the proliferation and cytokine production of CD4(+)CD25(-) responder T cells. The suppression requires cell-cell-contact and/or production of inhibitory cytokines like IL-10 or TGF-β. The current knowledge about the regulation of Treg suppressive function is limited. Toll-like receptors (TLR) are widely expressed in the innate immune system. They recognize conserved microbial ligands such as lipopolysaccharide, bacterial lipopeptides or viral and bacterial RNA and DNA. TLR play an essential role in innate immune responses and in the initiation of adaptive immune responses. However, certain TLR are also expressed in T lymphocytes, and the respective ligands can directly modulate T cell function. TLR2, TLR3, TLR5 and TLR9 act as costimulatory receptors to enhance proliferation and/or cytokine production of T-cell receptor-stimulated T lymphocytes. In addition, TLR2, TLR5 and TLR8 modulate the suppressive activity of naturally occurring CD4(+)CD25(high) Treg. The direct responsiveness of T lymphocytes to TLR ligands offers new perspectives for the immunotherapeutic manipulation of T cell responses. In this article we will discuss the regulation of Treg and other T cell subsets by TLR ligands.     

Regulation of human natural killer cell migration and proliferation by the exodus subfamily of CC chemokines

Natural killer (NK) cells play an important role in innate and adaptive immune responses to obligate intracellular pathogens. Nevertheless, the regulation of NK cell trafficking and migration to inflammatory sites is poorly understood. Exodus-1/MIP-3alpha/LARC, Exodus-2/6Ckine/SLC, and Exodus-3/MIP-3beta/ELC/CKbeta-11 are CC chemokines that share a unique aspartate-cysteine-cysteine-leucine motif near their amino terminus and preferentially stimulate the migration of T lymphocytes. The effects of Exodus chemokines on human NK cells were examined. Exodus-1, -2, and -3 did not induce detectable chemotaxis of resting peripheral blood NK cells. In contrast, Exodus-2 and -3 stimulated migration of polyclonal activated peripheral blood NK cells in a dose-dependent fashion. Exodus-2 and -3 also induced dose-dependent chemotaxis of NKL, an IL-2-dependent human NK cell line. Results of modified checkerboard assays indicate that migration of NKL cells in response to Exodus-2 and -3 represents true chemotaxis and not simply chemokinesis. Exodus-1, -2, and -3 did not induce NK cell proliferation in the absence of other stimuli. Nevertheless, Exodus-2 and -3 significantly augmented IL-2-induced proliferation of normal human CD56(dim) NK cells. In contrast, Exodus-1, -2, and -3 did not affect the cytolytic activity of resting or activated peripheral blood NK cells. Expression of message for CCR7, a shared receptor for Exodus-2 and -3, was detected in activated polyclonal NK cells and NKL cells but not resting NK cells. Taken together, these results indicate that Exodus-2 and -3 can participate in the recruitment and proliferation of activated NK cells. Exodus-2 and -3 may regulate interactions between T cells and NK cells that are crucial for the generation of optimal immune responses.     

Functions of IL-15 in anti-viral immunity: Multiplicity and variety

An effective immune response to an invading viral pathogen requires the combined actions of both innate and adaptive immune cells. For example, NK cells and cytotoxic CD8 T cells are capable of the direct engagement of infected cells and the mediation of antiviral responses. Both NK and CD8 T cells depend on common gamma chain (γc) cytokine signals for their development and homeostasis. The γc cytokine IL-15 is very well characterized for its role in promoting the development and homeostasis of NK cells and CD8 T cells, but emerging literature suggests that IL-15 mediates the anti-viral responses of these cell populations during an active immune response. Both NK cells and CD8 T cells must become activated, migrate to sites of infection, survive at those sites, and expand in order to maximally exert effector functions, and IL-15 can modulate each of these processes. This review focuses on the functions of IL-15 in the regulation of multiple aspects of NK and CD8 T cell biology, investigates the mechanisms by which IL-15 may exert such diverse functions, and discusses how these different facets of IL-15 biology may be therapeutically exploited to combat viral diseases.     

Antigen nonspecific suppression of T cell responses by activated stimulation-refractory CD4+ T cells

Several classes of anergic T cells are capable of suppressing naive T cell proliferation and thereby limiting immune responses. Activated T cells, although not anergic, are transiently refractory to restimulation with Ag. We examine in this study whether activated refractory murine T cells can also suppress naive T cell responses. We find that they can, and that they exhibit many of the suppressive properties of anergic T cells. The activated cells strongly diminish Ag-mediated T cell proliferation, an activity that correlates with their refractory period. Suppression is independent of APC numbers and requires cell contact or proximity. Naive T cells stimulated in the presence of activated refractory cells up-regulate CD25 and CD69, but fail to produce IL-2. The addition of IL-2 to culture medium, however, does not prevent the suppression, which is therefore not solely due to the absence of this growth factor. Persistence of the suppressor cells is also not essential. T cells stimulated in their presence and then isolated from them and cultured do not divide. The suppressive cells, however, do not confer a refractory or anergic state on the target T lymphocytes, which can fully respond to antigenic stimulation if removed from the suppressors. Our results therefore provide evidence that activated T cells act as transient suppressor cells, severely constraining bystander T cell stimulation and thereby restricting their response. These results have potentially broad implications for the development and regulation of immune responses.     

IL-21 is produced by NKT cells and modulates NKT cell activation and cytokine production

The common gamma-chain cytokine, IL-21, is produced by CD4(+) T cells and mediates potent effects on a variety of immune cells including NK, T, and B cells. NKT cells express the receptor for IL-21; however, the effect of this cytokine on NKT cell function has not been studied. We show that IL-21 on its own enhances survival of NKT cells in vitro, and IL-21 increases the proliferation of NKT cells in combination with IL-2 or IL-15, and particularly with the CD1d-restricted glycosphingolipid Ag alpha-galactosylceramide. Similar to its effects on NK cells, IL-21 enhances NKT cell granular morphology, including granzyme B expression, and some inhibitory NK receptors, including Ly49C/I and CD94. IL-21 also enhanced NKT cell cytokine production in response to anti-CD3/CD28 in vitro. Furthermore, NKT cells may be subject to autocrine IL-21-mediated stimulation because they are potent producers of this cytokine following in vitro stimulation via CD3 and CD28, particularly in conjunction with IL-12 or following in vivo stimulation with alpha-galactosylceramide. Indeed, NKT cells produced much higher levels of IL-21 than conventional CD4 T cells in this assay. This study demonstrates that NKT cells are potentially a major source of IL-21, and that IL-21 may be an important factor in NKT cell-mediated immune regulation, both in its effects on NK, T, and B cells, as well as direct effects on NKT cells themselves. The influence of IL-21 in NKT cell-dependent models of tumor rejection, microbial clearance, autoimmunity, and allergy should be the subject of future investigations.     

In vivo suppression of naive CD4 T cell responses by IL-2- and antigen-stimulated T lymphocytes in the absence of APC competition

After stimulation, T cells enter a transient refractory period, promoted by IL-2, during which they are resistant to re-stimulation. We previously demonstrated that these IL-2- and Ag-stimulated refractory T cells are able to suppress the Ag-induced proliferation of naive T cells in vitro. We show here that, after adoptive transfer, these T cells are also able to suppress naive T cell proliferation in vivo. More interestingly, potently suppressive T cells can be generated directly in vivo by stimulation with Ag and supplemental IL-2. The activity of the suppressive cells is dose dependent, and the suppressor and suppressed T cells need not be restricted to the same MHC or Ag. Similar to its role in promoting T cell-mediated suppression in vitro, IL-2 is critical for the induction of suppressive activity in activated T cells in vivo. Supplemental IL-2, however, cannot overcome the suppressive activity in target T cells, indicating that suppression is not mediated by competition for this cytokine. Although the activated T cells block naive T cell proliferation, the naive cells do engage Ag and up-regulate the CD25 and CD69 activation markers after stimulation. Therefore, activated T cells stimulated in the presence of IL-2 develop MHC- and Ag-unrestricted suppressive activity. These results provide a new mechanism for competition among CD4(+) T lymphocytes, in which initial waves of responding T cells may inhibit subsequently recruited naive T cells. They further suggest a novel negative feedback loop limiting the expansion of T cell responses that may be present during vigorous immune responses or after IL-2 immunotherapy.     

B lymphocyte stimulator regulates adaptive immune responses by directly promoting dendritic cell maturation

B lymphocyte stimulator (BLyS) is a well-known direct costimulator of adaptive immune cells, particularly B lineage cells. However, we have reported recently that BLyS is also able to activate monocytes. Other innate immune cells, such as dendritic cells (DCs), play a key role in the initiation of adaptive immune responses and the purpose of the current study was to assess whether there is a direct role for BLyS in modulating human DC functions. In this study, we show that BLyS induces DC activation and maturation. Thus, BLyS strongly induced up-regulation of surface costimulatory molecule expression and secretion of specific cytokines and chemokines in DCs. BLyS-stimulated DCs (BLyS-DCs) were also able to augment allogeneic CD4 T cell proliferation to a greater extent than control DCs. BLyS-DCs secreted elevated levels of the major Th1-polarizing cytokine, IL-12p70, and they promoted naive CD4 T cell differentiation into Th1 T cells. Regarding BLyS receptor expression, DCs primarily express cytoplasmic transmembrane activator and CAML interactor; however, low levels of cell surface transmembrane activator and CAML interactor are expressed as well. Collectively, our data suggest that BLyS may modulate adaptive immune cells indirectly by inducing DC maturation.