Relative efficiency of microglia, astrocytes, dendritic cells and B cells in naive CD4+ T cell priming and Th1/Th2 cell restimulation.

We have compared the efficiency of central nervous system and peripheral antigen-presenting cells (APC) in T cell priming and restimulation. OVA peptide 323 - 339-dependent activation of DO11.10 TCR-transgenic naive CD4+ and polarized Th1 or Th2 cells was assessed in the presence of microglia and astrocytes from the neonatal mouse brain as well as dendritic cells (DC) and B cells purified from adult mouse lymph nodes. DC were the most efficient in inducing naive T cell proliferation, IL-2 secretion and differentiation into Th1 cells, followed by IFN-gamma-preactivated microglia, large and small B cells. Astrocytes failed to activate naive T cells. IFN-gamma-pretreated microglia were as efficient as DC in the restimulation of Th1 cells, whereas IFN-gamma-pretreated astrocytes, large and small B cells were much less efficient. Conversely, Th2 cells were efficiently restimulated by all the APC types examined. During T cell priming, DC secreted more IL-12 than microglia but similar amounts of IL-12 were secreted by the two cell types upon interaction with Th1 cells. The hierarchy of APC established in this study indicates that DC and microglia are the most efficient in the stimulation of naive CD4(+) T cells and in the restimulation of Th1 cells, suggesting that activated microglia may effectively contribute to Th1 responses leading to central nervous system inflammation and tissue damage. These potentially pathogenic responses could be counteracted by the high efficiency of astrocytes as well as microglia in restimulating Th2 cells.     

Role of antigen-presenting cells in the polarized development of helper T cell subsets: evidence for differential cytokine production by Th0 cells in response to antigen presentation by B cells and macrophages

Immune challenges can elicit polarized responses skewed towards the development of T helper type 1 (Th1) or Th2 T cell subsets. To determine if distinct antigen-presenting cells (APC) populations might selectively influence Th subset development, we studied the role of two key APC populations, B cells and macrophages, in the differentiation of effector Th populations from naive precursor Th in vitro. Antigen (Ag)-specific, naive CD4⁺ T cells were enriched from a mouse strain, AND, bearing a transgenic α/β T cell receptor (TCR) encoding reactivity with pigeon cytochrome c peptide 88-104. Peptide Ag was used throughout these studies so that differences in the uptake and processing by the two APC populations would not influence the results. Both APC populations, activated B cells and bone marrow-derived macrophages, supported the development of effector Th having the capacity to secrete high levels of cytokines when restimulated. Regardless of APC population present during effector development, exogenous interferon-γ (IFN-γ) and interleukin-4 (IL-4) had dominant effects on Th subset development. Thus, with both APC populations, effector Th generated in the presence of IFN-γ acquired a Th1-type cytokine profile, Th generated with IL-4 acquired a Th2-type cytokine profile, and Th generated without IFN-γ or IL-4 acquired a Th0-type cytokine profile. B cells and macrophages also had equivalent APC function in the restimulation of Th1 and Th2-like effectors, since only minor differences in cytokine production were noted for these effector populations when restimulated with the two APC populations. However, in 8 of 19 experiments, the Th0-like effector population generated in the presence of IL-2 differentially responded to restimulation with B cells and macrophages, secreting significantly more IFN-γ when restimulated with B cells, and significantly more IL-4 when restimulated with macrophages. We also found that Th effector populations recultured in IFN-γ or IL-4 assumed a more Th1 or Th2-like phenotype, respectively, regardless of their initial cytokine profile. We conclude that through a subtle capacity to skew cytokine production by a Th0 subset, different APC may selectively influence Th subset development under conditions of prolonged or chronic stimulation in an autocrine fashion.     

Regulation of T helper cell differentiation in vivo by soluble and membrane proteins provided by antigen-presenting cells

The aim of this study was to test whether the nature of the antigen-presenting cell (APC) can influence the Th1 / Th2 balance in vivo. Our data show that dendritic cells (DC), pulsed extra corporeally with antigen, induced the development of cells secreting IL-2, IFN-γ and IL-4 upon antigen rechallenge in vitro. Priming with peritoneal macrophages sensitized cells that produced IL-4 but not IFN-γ. To identify the factors involved in T helper development, mice were primed with APC with or without treatment with neutralizing antibodies to co-stimulatory molecules or cytokines. Our results indicate that priming with DC or macrophages is strictly dependent on the CD28-CTLA4/ B7 interaction. Of note, CD86 provides the initial signal to induce naive T cells to become IL-4 producers, whereas CD80 is a more neutral differentiation signal. IL-12, released by the DC, appears as a potent and obligatory inducer of differentiation for IFN-γ-producing cells. IL-6, although produced by both APC populations, is necessary to direct activation of the Th2-type response by macrophages but not by DC.     

Similar co-stimulation requirements of CD4+ and CD8+ primary T helper cells: role of IL-1 and IL-6 in inducing IL-2 secretion and subsequent proliferation.

Primary murine CD4+ and CD8+ T helper (Th) cells provide help for various immune responses by secreting lymphokines which activate effector cells. The purpose of the present study was to investigate the co-stimulatory signals that, together with T cell receptor (TCR) cross-linking, induce phenotypically distinct primary Th cells to secrete IL-2 and proliferate. We isolated highly purified populations of primary CD4+ or CD8+ T cells and stimulated them in vitro with platebound anti-CD3 mAb. TCR cross-linking by anti-CD3 mAb induced both IL-2 receptor expression and responsiveness to exogenous IL-2, but was not sufficient to induce either IL-2 secretion or T cell proliferation. Rather, for both CD4+ and CD8+ primary Th cells, IL-2 secretion and proliferation required both TCR cross-linking and antigen presenting cell (APC)-derived co-stimulatory signals. Based on G-10 adherence and sensitivity to gamma-irradiation, the APC populations able to induce primary CD4+ Th cells and primary CD8+ Th cells to secrete IL-2 were indistinguishable. In addition, we found that either IL-1 or IL-6 could replace the requirement for APC-derived co-stimulatory signals for IL-2 secretion and proliferation by both primary CD4+ Th cells and primary CD8+ Th cells. Thus, the present study has examined and compared the co-stimulatory requirements of rigorously purified subsets of IL-2-secreting primary CD4+ and primary CD8+ T cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Availability of antigen-presenting cells can determine the extent of CD4 effector expansion and priming for secretion of Th2 cytokines in vivo

Like dendritic cells (DC), activated B cells are effective antigen-presenting cells (APC) for na?ve CD4 cells due to their expression of MHC class II and multiple costimulatory molecules. We showed previously that CD4 cells primed in B cell-deficient micro MT) mice undergo more limited expansion than in normal animals after immunization with keyhole limpet hemocyanin. Here we report that in the absence of B cells, priming of effectors with the capacity to produce the Th2 cytokines, IL-4, IL-5 and IL-13, was profoundly reduced whereas the development of effectors that secrete the Th1 cytokine IFN-gamma was much less affected. A blockade of IL-12 reduced priming of IFN-gamma-secreting effectors but did not reverse the IL-4, IL-5, or IL-13 deficiency of the response. CD4 cell expansion and priming for Th2 cytokines in micro MT mice was reconstituted by adoptive transfer of activated splenic B cells, which were present throughout the primary response. However, transfer of splenic DC from either control or micro MT mice also supported development of Th2 cytokine responses, indicating that an APC deficit rather than a unique contribution of B cells accounted for diminished effector priming. We conclude that CD4 cell expansion must be sustained via APC for the development of Th2 cytokine-secreting effectors in vivo and that in responses to protein antigen, B cells can be a crucial population to serve in this role. The results suggest that the level of APC engagement can not only determine the extent of effector expansion, but also the overall Th1/Th2 cytokine balance.     

Augmentation of naive,Th1 and Th2 effector CD4 Responses by IL-6, IL-1 and TNF

The role of antigen-presenting cell (APC)-derived cytokines in T cell activation is still controversial. Highly purified CD4 T cell populations of the naive and short-term Th1 and Th2 effector subsets were examined. Stimulation from anti-CD3 in the absence of APC was used to analyze directly T occurring cell-mediated effects, and the requirement for co-signaling was addressed using anti-CD28. Exogenous IL-6, IL-1 and TNF each enhanced proliferation and IL-2 secretion from naive cells, although IL-6 was most active in this regard. Peak responses, however, were obtained with IL-1 or TNF in combination with IL-6 resulting in up to 11-fold increases in IL-2 secretion. Enhanced naive T cell responses were only observed with anti-CD3 and anti-CD28, suggesting that co-signaling through surface-bound receptors was required to initiate IL-2 production. Although the cytokines enhanced naive activation, little effect was seen on differentiation into effector populations. IL-6 alone, or in combination, partially suppressed effectors secreting IFN-γ, but did not promote generation of effectors secreting IL-4. In contrast to reports on cloned cell lines, IL-6, TNF and IL-1 had enhancing activities on all cytokines elicited from already generated Th1 and Th2 effector populations. Again combinations of IL-6, TNF and IL-1 were most effective and generally required CD28 signaling. Induced responses with preexisting effector cells were far less than with naive cells and predominantly directed at augmenting IFN-γ and IL-5 secretion rather than IL-2 and IL-4. These studies show that APC-derived cytokines can promote T cell responses directly but largely after co-stimulation from accessory molecule co-receptors, that the effect is not specific for one T cell subset or cytokine, and that the naive T cell is the main target of action.     

Th1 cells induce and Th2 inhibit antigen-dependent IL-12 secretion by dendritic cells

Dendritic cells are the most relevant antigen-presenting cells (APC) for presentation of antigens administered in adjuvant to CD4⁺ T cells. Upon interaction with antigen-specific T cells, dendritic cells (DC) expressing appropriate peptide-MHC class II complexes secrete IL-12, a cytokine that drives Th1 cell development. To analyze the T cell-mediated regulation of IL-12 secretion by DC, we have examined their capacity to secrete IL-12 in response to stimulation by antigen-specific Th1 and Th2 DO11.10 TCR-transgenic cells. These cells do not differ either in TCR clonotype or CD40 ligand (CD40L) expression. Interaction with antigen-specific Th1, but not Th2 cells, induces IL-12 p40 and p75 secretion by DC. The induction of IL-12 production by Th1 cells does not depend on their IFN-γ secretion, but requires direct cell-cell contact mediated by peptide/MHC class II-TCR and CD40-CD40L interactions. Th2 cells not only fail to induce IL-12 secretion, but they inhibit its induction by Th1 cells. Unlike stimulation by Th1, inhibition of IL-12 production by Th2 cells is mediated by soluble molecules, as demonstrated by transwell cultures. Among Th2-derived cytokines, IL-10, but not IL-4 inhibit Th1-driven IL-12 secretion. IL-10 produced by Th2 cells appears to be solely responsible for the inhibition of Th1-induced IL-12 secretion, but it does not account for the failure of Th2 cells to induce IL-12 production by DC. Collectively, these results demonstrate that Th1 cells up-regulate IL-12 production by DC via IFN-γ-independent cognate interaction, whereas this is inhibited by Th2-derived IL-10. The inhibition of Th1-induced IL-12 production by Th2 cells with the same antigen specificity represents a novel mechanism driving the polarization of CD4⁺ T cell responses.     

Glucocorticoids drive human CD8(+) T cell differentiation towards a phenotype with high IL-10 and reduced IL-4, IL-5 and IL-13 production

Glucocorticoids are highly effective in the treatment of allergy and asthma and inhibit the synthesis of IL-4, IL-5 and IL-13 by disease-promoting CD4(+) Th2 cells. CD8(+) T cells also synthesize these cytokines, and the aim of this study was to investigate how glucocorticoids effect cytokine production by these cells. When CD8(+) T cells are stimulated with anti-CD3 and IL-2 plus IL-4 or dexamethasone, production of the anti-inflammatory cytokine IL-10 is low in both primary and secondary cultures restimulated with anti-CD3 and IL-2 alone. However, when both are present, a synergistic effect on IL-10 synthesis is observed. The additional presence of antigen-presenting cells (APC) in the priming culture maintains IL-10 levels, but inhibits IL-4 and IL-5 production. CD4(+) T cells develop a similar glucocorticoid-induced phenotype. These cells demonstrate regulatory activity and inhibit CD4(+) T cell activation in an IL-10-dependent manner. Earlier reports show glucocorticoids promote a Th2 phenotype by effects on purified naive T cells or pretreatment of APC. This study demonstrates, more critically, that when APC are present, glucocorticoids induce CD4 and CD8 T cell populations synthesizing high levels of IL-10, but greatly reduced amounts of disease-promoting IL-4 and IL-5.     

The role of antigen-presenting cells and interleukin-12 in the priming of antigen-specific CD4+ T cells by immune stimulating complexes

Immune stimulating complexes (ISCOMs) containing the saponin adjuvant Quil A are vaccine adjuvants that promote a wide range of immune responses in vivo, including delayed-type hypersensitivity (DTH) and the secretion of both T helper 1 (Th1) and Th2 cytokines. However, the antigen-presenting cell (APC) responsible for the induction of these responses has not been characterized. Here we have investigated the role of dendritic cells (DC), macrophages (Mφ) and B cells in the priming of antigen-specific CD4⁺ T cells in vitro by ISCOMs containing ovalbumin (OVA). OVA ISCOMs pulsed bone marrow (BM)-derived DC but not BM Mφ, nor naïve B cells prime resting antigen-specific CD4⁺ T cells, and this response is greatly enhanced if DC are activated with lipopolysaccharide (LPS). Of the APC found in the spleen, only DC had the capacity to prime resting antigen specific CD4⁺ T cells following exposure to OVA ISCOMs in vitro, while Mφ and B cells were ineffective. DC, but not B cells purified from the draining lymph nodes of mice immunized with OVA ISCOMs also primed resting antigen-specific CD4⁺ T cells in vitro, suggesting that DC are also critical in vivo. Using DC and T cells from interleukin (IL)-12 p40^−/− mice, we also identified a crucial role for IL-12 in the priming of optimal CD4⁺ T cell responses by OVA ISCOMs. We suggest that DC are the principal APC responsible for the priming of CD4⁺ T cells by ISCOMs in vivo and that directed targeting of these vectors to DC may enhance their efficancy as vaccine adjuvants.     

IL-15-induced human DC efficiently prime melanoma-specific naive CD8+ T cells to differentiate into CTL

Monocytes differentiate into dendritic cells (DC) in response to GM-CSF combined with other cytokines including IL-4 and IL-15. Here, we show that IL15-DC are efficient in priming naive CD8+ T cells to differentiate into melanoma antigen-specific cytotoxic T lymphocytes (CTL). While both melanoma peptide-pulsed IL15-DC and IL4-DC expand high-precursor frequency MART-1-specific CD8+ T cells after two stimulations in vitro, IL15-DC require much lower peptide concentration for priming. IL15-DC are more efficient in expanding gp100-specific CD8+ T cells and can expand CD8+ T cells specific for Tyrosinase and MAGE-3. CTL primed by IL15-DC are superior in their function as demonstrated by (i) higher IFN-gamma secretion, (ii) higher expression of Granzyme B and Perforin, and (iii) higher killing of allogeneic melanoma cell lines, most particularly the HLA-A*0201+ Sk-Mel-24 melanoma cells that are resistant to killing by CD8+ T cells primed with IL4-DC. Supernatants of the sonicated cells demonstrate unique expression of IL-1, IL-8 and IL-15. Therefore, membrane-bound IL-15 might contribute to enhanced priming by IL15-DC. Thus, IL-15 induces myeloid DC that are efficient in priming and maturation of melanoma antigen-specific CTL.     

The mast cell mediator PGD2 suppresses IL-12 release by dendritic cells leading to Th2 polarized immune responses in vivo

Dendritic cells (DC) and mast cells (MC) are colocalized in superficial organs such as the skin. Both cell types recognize and respond to pathogens. DC capture and transport antigens to the draining lymph node for CD4+ T cell priming and T helper 1 (Th1) or Th2 polarization. As MC are mainly associated with Th2 responses, DC-MC interactions may favor Th2 priming by DC. Here, we show the role of different MC mediators on IL-12 and IL-10 production by DC. While histamine, leukotriene C4, heparin and chondroitin sulfate A had little and unspecific effects on the cytokine production, prostaglandin D2 (PGD2) downregulated IL-10, IL-12p70 and p40. After subcutaneous (s.c.) injection of ovalbumin (OVA)-loaded, lipopolysaccharide (LPS)-matured DC into Th1-prone C57BL/6 mice, the levels of IFN-gamma produced by Th1 cells were decreased while IL-4 production remained low. When TNF-matured DC were pretreated with PGD2, loaded with the endotoxin-free antigen KLH and injected s.c. into Th2-prone BALB/c mice, we found a dose- and time-dependent upregulation of IL-4 and downregulation of IFN-gamma by T cells. Together, MC-derived PGD2 instructs DC to polarize CD4+ T cells towards Th2 responses. As a consequence, such a DC-MC cooperation may contribute to the maintenance of Th2 responses in allergic patients.     

Dendritic cells retrovirally overexpressing IL-12 induce strong Th1 responses to inhaled antigen in the lung but fail to revert established Th2 sensitization

It has been postulated that low-level interleukin (IL)-12 production of antigen-presenting cells is associated with the risk of developing atopic asthma. To study the relationship between IL-12 production capacity of dendritic cells (DCs) and development of T helper type 2 (Th2) responses in the lung, we genetically engineered DCs to constutively overexpress bioactive IL-12. Retrovirally mediated overexpression of IL-12 in DCs strongly polarized naive ovalbumin (OVA)-specific CD4+ T cells toward Th1 effector cells in vitro. After intratracheal injection, OVA-pulsed IL-12-overexpressing DCs failed to induce Th2 responses in vivo and no longer primed mice for Th2-dependent eosinophilic airway inflammation upon OVA aerosol challenge, readily observed in mice immunized with sham-transfected, OVA-pulsed DCs. Analysis of a panel of cytokines and chemokines in the lung demonstrated that the lack of Th2 sensitization was accompanied by increased production of the Th1 cytokine interferon-gamma (IFN-gamma), chemokines induced by IFN-gamma, and the immunoregulatory cytokine IL-10. When Th2 priming was induced using OVA/alum prior to intratracheal DC administration, DCs constitutively expressing IL-12 were no longer capable of preventing eosinophilic airway inflammation and even enhanced it. These data show directly that high-level expression of IL-12 in DCs prevents the development of Th2 sensitization. Enhancing IL-12 production in DCs should be seen as a primary prevention strategy for atopic disorders. Enhancing IL-12 production in DCs is less likely to be of benefit in already Th2-sensitized individuals.     

UVB-irradiated dendritic cells are impaired in their APC function and tolerize primed Th1 cells but not naive CD4+ T cells

We have shown that low-dose UVB radiation converts Langerhans cells (LC) from immunogenic to tolerogenic APC. Therefore, we questioned whether low-dose UVB irradiation of bone marrow-derived dendritic cells (DC) alters their APC function, thereby inducing tolerance in T cells. To address this issue, cocultures of DC; and naive, allogeneic T cells; na?ve, OVA-specific TCR-transgenic T cells from DO11.10 mice; or primed, antigen-specific T cells using the Th1 clone AE7 were analyzed. First, we found low-dose UVB-irradiated DC (UVB-DC) to dose-dependently (50-200 J/m2) inhibit T-cell proliferation of naive and primed T cells. In addition, supernatants harvested from cocultures of UVB-DC and naive T cells showed markedly reduced levels of IL-2 and IFN-gamma and to a lesser degree of IL-4 and IL-10, suggesting a preferential down-regulation of Th1 responses by UVB-DC. FACS analysis of UVB-DC revealed no changes in surface expression of MHC, costimulatory, and adhesion molecules. To test tolerance induction, allo- or antigen-specific T cells isolated from cocultures with unirradiated DC and UVB-DC were restimulated with unirradiated DC or IL-2. It is interesting that UVB-DC induced antigen-specific tolerance in the Th1 clone AE7. In contrast, UVB-DC induced a partial inhibition of allogeneic T-cell proliferation but no tolerance with similar unresponsiveness to restimulation with IL-2 and unirradiated DC irrespective of their haplotype. Similar observations were made when naive, TCR-transgenic T cells from DO11.10 mice were used. In conclusion, UVB-DC are impaired in their APC function and tolerize the primed antigen-specific Th1 clone AE7 but not naive allo- or OVA-specific T cells.     

Prostaglandin E2 primes naive T cells for the production of anti-inflammatory cytokines

In addition to their capacity to induce pain, vasodilatation and fever, prostaglandins E (PGE) exert anti-inflammatory activities by inhibiting the release of pro-inflammatory cytokines by macrophages and T cells, and by increasing interleukin (IL)-10 production by macrophages. We here report that PGE₂, the major arachidonic acid metabolite released by antigen-presenting cells (APC), primes naive human T cells for enhanced production of anti-inflammatory cytokines and inhibition of pro-inflammatory cytokines. Unfractionated as well as CD45RO^?CD31⁺ sort-purified neonatal CD4 T cells acquire the capacity to produce a large spectrum of cytokines after priming with anti-CD3 and anti-CD28 monoclonal antibodies (mAb), in the absence of both APC and exogenous cytokines. PGE₂ primes naive T cells in a dose-dependent fashion for production of high levels of IL-4, IL-10 and IL-13, and very low levels of IL-2, interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and TNF-β. PGE₂ does not significantly increase IL-4 production in priming cultures, whereas it suppresses IL-2 and IFN-γ. Addition of a neutralizing mAb to IL-4 receptor in primary cultures, supplemented or not with PGE₂, prevents the development of IL-4-producing cells but does not abolish the effects of PGE₂ on IL-10 and IL-13 as well as T helper (Th)1-associated cytokines. Addition of exogenous IL-2 in primary cultures does not alter the effects of PGE₂ on naive T cell maturation. Thus PGE₂ does not act by increasing IL-4 production in priming cultures, and its effects are partly IL-4 independent and largely IL-2 independent. Together with the recent demonstration that PGE₂ suppresses IL-12 production, our results strongly suggest that this endogenously produced molecule may play a significant role in Th subset development and that its stable analogs may be considered for the treatment of Th1-mediated inflammatory diseases.     

Environmental pollutant tributyltin promotes Th2 polarization and exacerbates airway inflammation

It has been shown that a relatively high dose of tributyltin (TBT), which is recognized as a particularly notable environmental pollutant, exerts immunotoxic effects such as thymic atrophy via induction of T cell apoptosis. However, the effect of low doses of TBT on the immune responses remains unknown. Here we show that environmentally relevant doses of TBT promoted strong Th2 polarization via suppression and augmentation of Th1 and Th2 development, respectively, from naive CD4(+) T cells primed with anti-CD3 and splenic antigen-presenting cells (APC). TBT-induced Th2 polarization was indirect, working through APC via suppression of IL-12 production by macrophages/DC and the augmentation of IL-10 production by B cells. Th2 polarization was also induced in mice treated with TBT and immunized with OVA or infected with Nippostrongylus brasiliensis. Furthermore, airway inflammation in mice sensitized and challenged with OVA was exacerbated by the administration of TBT with concomitant augmentation of Th2-type immunity. Our results highlight the fact that an important environmental pollutant TBT may present significant risk for the induction of allergic diseases via promotion of Th2 polarization.     

Naive human T cells can be a source of IL-4 during primary immune responses

IL-4 plays a key role in driving the differentiation of CD4+ Th precursors into Th2 cells, both in mice and in humans. The source of IL-4 during primary immune responses is, however, still debated. When IL-4 consumption in in vitro T cell cultures was blocked with a MoAb to the IL-4 receptor alpha-chain (IL-4Ralpha), it became evident that freshly isolated naive (CD45RO-) CD4+ T cells from adults or cord blood produce IL-4 upon activation with anti-CD3 and CD80. IL-4 production by naive T cells is strictly IL-2-dependent. Endogenous IL-4 activity in naive CD4+ T cell cultures modulates the production of interferon-gamma (IFN-gamma) on the one hand and IL-5 and IL-13 on the other hand in opposite directions, and it is partly responsible for the low IFN-gamma production by cord blood T cells. Comparison of the ratio of IL-4/IFN-gamma in supernatants of T cell cultures reveals a skewing towards IL-4 production by cord blood T cells, while naive T cells from (non-atopic) adults predominantly produce IFN-gamma. We conclude that CD4+ naive T cells can produce IL-4 without the need for Th2 differentiation, and therefore that they can be the initial source of IL-4 required at the time of priming for T cell differentiation into Th2 cells.