Instruction of naive CD4+ T cells by polarized CD4+ T cells within dendritic cell clusters

Cooperation between CD4(+) T cells can enhance the response and modulate the cytokine profile, and defining these parameters has become a major issue for multivalent-vaccine strategies.We explored cooperation using adoptive transfer of two populations of TCR transgenic T cells of different specificity. One was transferred without prior activation, whereas the second was activated for five days by antigen stimulation under polarizing culture conditions. Both populations were transferred into a single adoptive host and then primed by particle-mediated DNA delivery. Polarized Th1 cells (inducers) raised the frequency of IFN-gamma(+) cells within a naive (target) population, whereas Th2 inducers raised the frequency of IL-4(+) and reduced that of IL-2(+) cells. These effects were obtained when the genes for both antigens were on the same particle, favoring presentation by the same dendritic cell, but not when on different particles delivered to different dendritic cells. Autonomy of DC clusters allows linked sets of antigens (e.g. from a single pathogen) to maintain cytokine bias, but allows other independent responses, each with their own set of autonomous clusters.     

Multiple survival signals are delivered by dendritic cells to naive CD4+ T cells

The molecular mechanisms by which dendritic cells (DC) favor naive T cell survival in mice have been examined in co-cultures of DC and naive CD4+ T cells. Naive T cells can survive in the presence of IL-4 or IL-7, but DC-induced T cell survival requires direct cell-cell interactions and does not seem to be mediated by these or other soluble factors. Classical MHC II molecules on DC are not necessary for T cell survival as long as hybrid AalphaEbeta MHC class II molecules are present. In the total absence of MHC II molecules on DC, T cell survival is reduced by half, and CD3zeta phosphorylation fully disappears. These results contrast with the classical view that naive T cell survival is associated with CD3zeta phosphorylation and depends mostly on IL-7 and MHC-TCR interactions. We demonstrate that DC-induced T cell survival is a multi-factorial process that also involves CD28, LFA-1 and another (as yet undefined) surface molecule that requires the activity of src (but not phosphatidylinositol-3-) kinase.     

Interleukin-4 selectively inhibits interleukin-2 secretion by lipopolysaccharide-activated dendritic cells

Dendritic cells (DCs) generated in vitro from bone marrow precursors using granulocyte-macrophage colony-stimulating factor (GM-CSF) secrete interleukin-2 (IL-2) upon activation, an event probably associated to the initiation of adaptive immune responses. Additionally, they produce IL-12, a cytokine related to T-cell polarization. To analyse the effect of IL-4 on DC differentiation and function, we assessed the capacity of murine bone marrow dendritic cells (BMDCs) differentiated with GM-CSF in the presence or absence of IL-4 to produce IL-2 and IL-12 upon lipopolysaccharide (LPS) activation. We found that although IL-4 enhanced DC IL-12p70 production, it strongly impaired IL-2 secretion by BMDCs. This inhibition, which depends on the presence of IL-4 during LPS activation, is DC specific, as IL-4 did not affect IL-2 secretion by T cells. Interestingly, inhibition of DC IL-2 production did not prevent DC priming of T lymphocytes. These results illustrate a new putative role for IL-4 on the regulation of the immune response and should help clarify the controversial reports on the effect of IL-4 on DCs.     

Inhibition of phosphoantigen-mediated gammadelta T-cell proliferation by CD4+ CD25+ FoxP3+ regulatory T cells

Tumour growth promotes the expansion of CD4(+) CD25(+) FoxP3(+) regulatory T cells (Tregs) which suppress various arms of immune responses and might therefore contribute to tumour immunosurveillance. In this study, we found an inverse correlation between circulating Treg frequencies and phosphoantigen-induced gammadelta T-cell proliferation in cancer patients, which prompted us to address the role of Tregs in controlling the gammadelta T-cell arm of innate immune responses. In vitro, human Treg-peripheral blood mononuclear cell (PBMC) co-cultures strongly inhibited phosphoantigen-induced proliferation of gammadelta T cells and depletion of Tregs restored the impaired phosphoantigen-induced gammadelta T-cell proliferation of cancer patients. Tregs did not suppress other effector functions of gammadelta T cells such as cytokine production or cytotoxicity. Our experiments indicate that Tregs do not mediate their suppressive activity via a cell-cell contact-dependent mechanism, but rather secrete a soluble non-proteinaceous factor, which is independent of known soluble factors interacting with amino acid depletion (e.g. arginase-diminished arginine and indolamine 2,3-dioxygenase-diminished tryptophan) or nitric oxide (NO) production. However, the proliferative activity of alphabeta T cells was not affected by this cell-cell contact-independent suppressive activity induced by Tregs. In conclusion, these findings indicate a potential new mechanism by which Tregs can specifically suppress gammadelta T cells and highlight the strategy of combining Treg inhibition with subsequent gammadelta T-cell activation to enhance gammadelta T cell-mediated immunotherapy.     

CD25+ CD4+ T cells compete with naive CD4+ T cells for IL-2 and exploit it for the induction of IL-10 production

Maintenance of homeostasis in the immune system involves competition for resources between T lymphocytes, which avoids the development of immune pathology seen in lymphopenic mice. CD25+ CD4+ T cells are important for homeostasis, but there is as yet no consensus on their mechanisms of action. Although CD25+ CD4+ T cells cause substantial down-regulation of IL-2 mRNA in responder T cells in an in vitro co-culture system, the presence of IL-protein can be demonstrated by intracellular staining. As a consequence of competition for IL-2, CD25+ CD4+ T cells further up-regulate the IL-2R alpha chain (CD25), a process that is strictly dependent on IL-2, whereas responder T cells fail to up-regulate CD25. Similarly, adoptive transfer into lymphopenic mice showed that CD25+ CD4+ T cells interfere with CD25 up-regulation on co-transferred naive T cells, while increasing their own CD25 levels. IL-2 sequestration by CD25+ CD4+ T cells is not a passive phenomenon but instead initiates--in conjunction with signals through the TCR--their differentiation to IL-10 production. Although IL-10 is not required for in vitro suppression, it is vital for the in vivo function of regulatory T cells. Our data provide a link explaining the apparent difference in regulatory mechanisms in vitro and in vivo.     

Inhibition of contact sensitivity in human CD4+ transgenic mice by human CD4-specific monoclonal antibodies: CD4+ T-cell depletion is not required

Clenoliximab and keliximab are monkey/human chimeric monoclonal antibodies (mAbs) of the immunoglobulin G4 (IgG4) and IgG1 isotypes, respectively, that recognize the same epitope on human CD4. The two mAbs possess identical idiotypes and exhibit equal affinities for CD4. Upon administration of these mAbs to mice that express a human CD4 transgene, but not mouse CD4 (HuCD4/Tg mice), clenoliximab and keliximab exhibited similar kinetics of binding to CD4, and induced the same degree of CD4 modulation from the cell surface, although only keliximab mediated CD4+ T-cell depletion. Epicutaneous sensitization and challenge of HuCD4/Tg mice with the hapten oxazolone resulted in a contact sensitivity response characterized by tissue swelling, and the presence of interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) in the local tissue. Administration of a single 2-mg dose of either clenoliximab or keliximab to HuCD4/Tg mice prior to sensitization significantly reduced post-challenge tissue swelling, and levels of IFN-gamma and IL-4, indicating that CD4+ T-cell depletion is not required for anti-CD4 mAb-mediated inhibition of contact sensitivity. Administration of either mAb prior to challenge failed to inhibit the contact sensitivity response, indicating differential sensitivity of the afferent and efferent phases of the response to inhibition by CD4-specific mAbs. Collectively, these data indicate that CD4 functions as a positive regulatory molecule in the contact sensitivity response.     

Viral dsRNA-activated human dendritic cells produce IL-27, which selectively promotes cytotoxicity in naive CD8+ T cells

Viral recognition programs DCs to express Signal 3 molecules that promote the differentiation of effector CD8(+) T cells. Besides IL-12, another DC-derived IL-12 family member, IL-27, has been reported to contribute herein, but its specific role is not well understood. Here, we show that whereas IL-12 potently induces inflammatory cytokines (i.e., IFN-γ and TNF-α, but not IL-2), IL-27 excels in inducing proliferation and a cytotoxic profile (GrB, cytotoxicity of target cells) in human na?ve CD8(+) T cells. Compared with bacterial cell-wall peptidoglycan, viral dsRNA-mimic poly (I:C) is superior in priming human BDCA1(+) peripheral blood DCs to produce IL-12 and IL-27, which promote inflammatory cytokines and a cytotoxic profile in differentiating CD8(+) T cells, respectively. These data support the concept that viral dsRNA-activated human DCs produce IL-27 to act as a specialized procytotoxic, antiviral cytokine in the development of effector CD8(+) T cells.     

Steady-state migrating intestinal dendritic cells induce potent inflammatory responses in naive CD4+T cells

Steady-state dendritic cells (DCs) migrating in the lymph from the intestine induce tolerance to harmless intestinal antigens, preventing inflammatory responses. To determine if such DCs are inherently tolerogenic we collected intestinal lymph DCs (L-DCs) by cannulation of the thoracic duct of rats after mesenteric lymphadenectomy, and examined their capacity to activate naive CD4⁺ lymphocytes in an allogeneic mixed leucocyte reaction. L-DCs stimulated strong proliferative responses, induced secretion of inflammatory cytokines including interferon-γ, and induced FoxP3-positive lymphocytes to divide. To determine if the activated CD4⁺ T cells had been tolerized, they were rested and restimulated with irradiated splenocytes. The restimulated CD4⁺ T cells again proliferated and secreted inflammatory cytokines. These data demonstrate that the DCs, which migrate from the intestine in the steady state, are paradoxically able to induce strong inflammatory responses from naive T cells, despite their role in the maintenance of oral tolerance.     

Aberrant induction of regulatory activity of CD4+CD25+ T cells by dendritic cells in HIV-infected persons with amebic liver abscess

To know why HIV-1-infected persons are particularly susceptible to amebic liver abscess (ALA), we investigated the role of CD4CD25 T cells in the susceptibility of HIV-1-infected persons to this disease. Herein we show, in early stage HIV-1-infected subjects, that CD4 T-cell responses to Entamoeba histolytica antigen (EhAg) were selectively impaired, especially in those with ALA. EhAg-specific CD4 T-cell responses were normalized by depletion of CD4CD25 cells or by addition of anti-cytotoxic T lymphocyte antigen 4 (CTLA4) antibody. Regulatory activity of CD4CD25 T cells to suppress the EhAg-specific CD4 T-cell response could be induced by EhAg-primed dendritic cells (DCs) in HIV-1-infected subjects, especially in those with ALA, but not in healthy controls. Exogenous Tat-incubated DCs derived from HIV-negative subjects also could upregulate CTLA4 expression on autologous CD4CD25 T cells and selectively suppress the EhAg-specific CD4 T-cell response. The results imply an interaction of the two pathogens: HIV-1, perhaps through the effect of Tat on DCs, may upregulate EhAg-specific regulatory T-cell activity to suppress T-cell response to E. histolytica, thus increasing the susceptibility to invasive amebiasis in even early-stage HIV-1-infected persons.     

Mouse lung CD103+ and CD11bhigh dendritic cells preferentially induce distinct CD4+ T-cell responses

Mouse lung dendritic cells (LDCs) have been recently shown to contain two major subpopulations: CD103(+) CD11b(low or negative) (CD103(+) LDCs) and CD103(-) CD11b(high) LDCs (CD11b(high) LDCs). Although several studies have demonstrated functional differences between them, it is unclear whether the subpopulations induce distinct T helper (Th) cell responses. The present study was conducted to examine whether CD103(+) and CD11b(high) LDCs preferentially generate different Th responses. Naive DO11.10 CD4(+) T cells were primed with CD103(+) or CD11b(high) LDCs obtained from normal BALB/c mice. The primed CD4(+) T cells were restimulated, and their cytokine secretions were assessed. The expression of intracellular cytokines and the mRNA levels of chemokine receptors were also measured. We found that the CD4(+) T cells primed with CD103(+) LDCs secreted significantly larger amounts of IFN-γ and IL-17A, whereas those primed with CD11b(high) LDCs released significantly higher levels of IL-4, IL-6, and IL-10. Intracellular cytokine assay showed that CD103(+) LDCs induced greater frequencies of CD4(+) T cells producing IFN-γ and IL-17A, whereas CD11b(high) LDCs were more efficient at inducing CD4(+) T cells producing IL-4 and IL-10. The mRNA levels of CXCR3 and CCR5, which are expressed preferentially in Th1 cells, were significantly higher in CD4(+) T cells primed with CD103(+) LDCs. The mRNA levels of CXCR4 and CCR4, which are expressed primarily in Th2 cells, were significantly greater in those primed with CD11b(high) LDCs. These data suggest that mouse CD103(+) LDCs predominantly elicit Th1 and Th17 responses, whereas CD11b(high) LDCs primarily provoke a Th2 response under the steady state.     

Type I interferon supports primary CD8+ T-cell responses to peptide-pulsed dendritic cells in the absence of CD4+ T-cell help

CD8(+) T-cell responses to non-pathogen, cell-associated antigens such as minor alloantigens or peptide-pulsed dendritic cells (DC) are usually strongly dependent on help from CD4(+) T cells. However, some studies have described help-independent primary CD8(+) T-cell responses to cell-associated antigens, using immunization strategies likely to trigger natural killer (NK) cell activation and inflammatory cytokine production. We asked whether NK cell activation by MHC I-deficient cells, or administration of inflammatory cytokines, could support CD4(+) T-cell help-independent primary responses to peptide-pulsed DC. Injection of MHC I-deficient cells cross-primed CD8(+) T-cell responses to the protein antigen ovalbumin (OVA) and the male antigen HY, but did not stimulate CD8(+) T-cell responses in CD4-depleted mice; hence NK cell stimulation by MHC I-deficient cells did not replace CD4(+) T-cell help in our experiments. Dendritic cells cultured with tumour necrosis factor-α (TNF-α) or type I interferon-α (IFN-α) also failed to prime CD8(+) T-cell responses in the absence of help. Injection of TNF-α increased lymph node cellularity, but did not generate help-independent CD8(+) T-cell responses. In contrast, CD4-depleted mice injected with IFN-α made substantial primary CD8(+) T-cell responses to peptide-pulsed DC. Mice deficient for the type I IFN receptor (IFNR1) made CD8(+) T-cell responses to IFNR1-deficient, peptide-pulsed DC; hence IFN-α does not appear to be a downstream mediator of CD4(+) T-cell help. We suggest that primary CD8(+) T-cell responses will become help-independent whenever endogenous IFN-α secretion is stimulated by tissue damage, infection, or autoimmune disease.     

CD5 monoclonal antibodies react with human peripheral blood dendritic cells.

CD5 monoclonal antibodies (MAbs) define a 67,000 kd monomeric glycoprotein expressed predominantly by thymocytes, mature T cells and a subpopulation of B cells. CD5 is believed to be an alternative signaling molecules capable of increasing the supply of second messengers and thereby altering the cellular response threshold to other activation stimuli. Human peripheral blood dendritic cells (PBDC) are a circulating component of the immune dendritic cell family, which also includes Langerhans' cells in epithelia and interdigitating cells in the T-cell domains of lymphoid tissues. PBDC comprise less than 1% of the peripheral blood mononuclear cell fraction. They are morphologically, immunophenotypically, and functionally distinct from monocytes. In this study, we report that at least a subpopulation of PBDC react with the anti-CD5 MAbs Leu-1 and UCHT2, which define the two major non-crossblocking CD5 epitopes. In contrast, Langerhans' cells, interdigitating cells, monocytes, and macrophages were uniformly CD5-. These findings suggest that PBDC can express the CD5 molecule. Furthermore, they define an additional feature of many enriched PBDC that distinguishes them from monocytes and certain other mononuclear leukocytes, and may provide insights into their activation pathways.     

Minimal activation of memory CD8+ T cell by tissue-derived dendritic cells favors the stimulation of naive CD8+ T cells

Of the many dendritic cell (DC) subsets, DCs expressing the monomorphic coreceptor CD8 alpha-chain (CD8alpha) are localized permanently in lymphoid organs, whereas 'tissue-derived DCs' remain in nonlymphoid tissues until they 'capture' antigen and then move to local lymph nodes. Here we show that after lung infection, both naive and memory CD8+ 'killer' T cells responded to influenza virus antigens presented by lymph node-resident CD8alpha+ DCs, but only naive cells responded to antigens presented by lung-derived DCs. This difference provides a mechanism for priming naive T cell responses in conditions in which robust memory predominates. Our findings have implications for immunity to pathogens that can mutate their T cell epitopes, such as influenza virus and human immunodeficiency virus, and challenge the long-held view that memory T cells have less-stringent requirements for activation than naive T cells have.     

Increased interleukin-10 production by ASC-deficient CD4+ T cells impairs bystander T-cell proliferation

Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is an important component of the inflammasome, functioning as an adaptor protein that facilitates the recruitment and activation of procaspases that in turn promote the maturation of interleukin-1β (IL-1β) and IL-18. Despite initial focus on the inflammatory properties of ASC there is emerging evidence that highlights the importance of ASC in facilitating adaptive immune responses. However, the cellular and molecular basis for the involvement of ASC in adaptive immunity remains largely unexplored. We have previously demonstrated that activated ASC-deficient T cells have dampened proliferative responses. We have therefore explored the underlying cellular mechanism(s) by which ASC regulates T-cell proliferation. We show that under activating conditions (anti-CD3/CD28 stimulation) in bulk T-cell cultures the presence of ASC(-/-) CD4(+) T cells is sufficient to suppress the proliferative responses of neighbouring T cells. Furthermore, ASC(-/-) CD4(+) T cells upon activation exhibit a suppressive cytokine profile, with elevated production of IL-10 and reduced secretion of T helper type 1 cytokines, interferon-γ and IL-2. This increase in IL-10 secretion within the activated ASC(-/-) CD4(+) T-cell compartment was not associated with a proportional increase in conventional Foxp3(+) regulatory T (Treg) cells. Interestingly, when equal numbers of fluorescence-activated cell sorted ASC(+/+) and ASC(-/-) Treg cells (CD4(+) CD44(intermediate/high) CD25(+)) were activated in vitro, the ASC(-/-) fraction produced significantly more IL-10 than their wild-type counterparts, suggesting that ASC(-/-) Treg cells have greater suppressive capacity. Collectively, these results imply that the ASC may influence the development and functioning of Treg cells.     

CD8alpha+ dendritic cells enhance the antigen-specific CD4+ T-cell response and accelerate development of collagen-induced arthritis

To investigate the role of CD8alpha(+) DCs in the development of collagen-induced arthritis (CIA). The immunogenic properties of CD8alpha(+) and CD8alpha(-) DC subsets were investigated by mixed-lymphocyte reaction and cytokine enzyme-linked immunoassay. CII-pulsed CD8alpha(+) DCs or CD8alpha(-) DCs with CD4(+) T cells from CIA mice were adoptively transferred onto the hind footpad of DBA mice. The onset of arthritis and the arthritis index were examined for 14 weeks after adoptive transfer. Expression of MHC-II and CD80 but not CD86 and CD40 was higher in CD8alpha(+) DCs than in CD8alpha(-) DCs from the spleens of CIA mice. Culturing CD8alpha(+) DCs with CD4(+) T cells significantly increased the proliferative response of CD4(+) T cells in the presence of CII. The production of interleukin (IL)-12p70, IL-17, interferon (IFN)-gamma, and tumor necrosis factor (TNF)-alpha was slightly increased in CD8alpha(+) DCs than in CD8alpha(-) DCs. DBA/1 mice that were adoptively transferred with CII-pulsed CD8alpha(+) DCs and CD4(+) T cells into the footpads showed accelerated onset of CIA compared to control group. By contrast, CD8alpha(-) DCs showed a partial inhibitory effect on CIA. These findings show that CD8alpha(+) DCs accelerated the onset of CIA when aoptively transferred with CD4(+) T cells and that CD8alpha(+) DCs provoke the development of CIA probably by stimulating the immune responses of CII-reactive CD4(+) T cells and by increasing the production of inflammatory cytokines.     

2E3, a new marker that selectively identifies porcine CD4+ naive T cells

We describe a novel antigen recognized by mAb 2E3 selectively expressed in the periphery by a subset of porcine CD4+ T cells. Both, CD4+CD8alpha- and CD4+CD8alphalow T cell subpopulations express this antigen. CD4+2E3+ T cells show phenotypical and functional characteristics of nai;ve cells. The majority of them are CD29low, CD45RAhigh, CD49dlow, CD11alow, CD18low, and SLA-II-. After mitogen activation CD4+2E3+ T cells express high levels of IL-2 mRNA, but only traces of IFN-gamma or IL-4 mRNA. Indeed a minor percentage of cells stained positive for IFN-gamma when assessed by flow cytometry. Moreover, CD4+2E3+ T cells did not proliferate in response to the recall antigen lysozyme, although they did efficiently to the mitogen ConA. By contrast, CD4+2E3- T cells show phenotypical and functional characteristics of primed cells. They express markers associated to a memory phenotype, respond to the recall antigen lysozyme, and produce high amounts of IFN-gamma and IL-4.     

IL-15 and dendritic cells induce proliferation of CD4+CD25+ regulatory T cells from peripheral blood

CD4(+)CD25(+) regulatory T cells (Tregs) have recently been the subject of intense research due to their strong immunosuppressive effect. Increasing evidence suggests that IL-15 plays an important role in Tregs biology. Nevertheless, the mechanism by which IL-15 performs this function remains to be fully elucidated. To address this question, we isolated Tregs from human peripheral blood, and utilized IL-15, dendritic cells (DCs), or DCs combined with IL-15, to examine the proliferation of Tregs and to explore related molecular mechanisms. Here, we show that IL-15 can induce the proliferation of Tregs in the presence of DCs. The induction is mediated by DCs presenting IL-15 in trans to Tregs. Simultaneously, DCs-derived IL-2, regulated by IL-15, may also play a supportive role. After IL-15 withdrawal, IL-15 trans-endosomal recycling in DCs contributes to the proliferation of Tregs. The activation of Akt, Erk1/2 and STAT(5), and the degradation of p27(kip1) may be involved in this process. These findings might explain the proliferation of Tregs in the absence of IL-2 in vivo and provide a novel method to achieve large-scale proliferation of Tregs in vitro in order to obtain cell numbers sufficient for immunotherapy.