CD4+ CD25+ regulatory T cells control T helper cell type 1 responses to foreign antigens induced by mature dendritic cells in vivo

Recent evidence suggests that in addition to their well known stimulatory properties, dendritic cells (DCs) may play a major role in peripheral tolerance. It is still unclear whether a distinct subtype or activation status of DC exists that promotes the differentiation of suppressor rather than effector T cells from naive precursors. In this work, we tested whether the naturally occurring CD4+ CD25+ regulatory T cells (Treg) may control immune responses induced by DCs in vivo. We characterized the immune response induced by adoptive transfer of antigen-pulsed mature DCs into mice depleted or not of CD25+ cells. We found that the development of major histocompatibility complex class I and II-restricted interferon gamma-producing cells was consistently enhanced in the absence of Treg. By contrast, T helper cell (Th)2 priming was down-regulated in the same conditions. This regulation was independent of interleukin 10 production by DCs. Of note, splenic DCs incubated in vitro with Toll-like receptor ligands (lipopolysaccharide or CpG) activated immune responses that remained sensitive to Treg function. Our data further show that mature DCs induced higher cytotoxic activity in CD25-depleted recipients as compared with untreated hosts. We conclude that Treg naturally exert a negative feedback mechanism on Th1-type responses induced by mature DCs in vivo.     

Human dendritic cells activated by TSLP and CD40L induce proallergic cytotoxic T cells

Human thymic stromal lymphopoietin (TSLP) is a novel epithelial cell-derived cytokine, which induces dendritic cell (DC)-mediated CD4+ T cell responses with a proallergic phenotype. Although the participation of CD8+ T cells in allergic inflammation is well documented, their functional properties as well as the pathways leading to their generation remain poorly understood. Here, we show that TSLP-activated CD11c+ DCs potently activate and expand naive CD8+ T cells, and induce their differentiation into interleukin (IL)-5 and IL-13-producing effectors exhibiting poor cytolytic activity. Additional CD40L triggering of TSLP-activated DCs induced CD8+ T cells with potent cytolytic activity, producing large amounts of interferon (IFN)-gamma, while retaining their capacity to produce IL-5 and IL-13. These data further support the role of TSLP as initial trigger of allergic T cell responses and suggest that CD40L-expressing cells may act in combination with TSLP to amplify and sustain pro-allergic responses and cause tissue damage by promoting the generation of IFN-gamma-producing cytotoxic effectors.     

Activation of natural killer T cells by alpha-galactosylceramide rapidly induces the full maturation of dendritic cells in vivo and thereby acts as an adjuvant for combined CD4 and CD8 T cell immunity to a coadministered protein

The maturation of dendritic cells (DCs) allows these antigen-presenting cells to initiate immunity. We pursued this concept in situ by studying the adjuvant action of alpha-galactosylceramide (alphaGalCer) in mice. A single i.v. injection of glycolipid induced the full maturation of splenic DCs, beginning within 4 h. Maturation was manifest by marked increases in costimulator and major histocompatibility complex class II expression, interferon (IFN)-gamma production, and stimulation of the mixed leukocyte reaction. These changes were not induced directly by alphaGalCer but required natural killer T (NKT) cells acting independently of the MyD88 adaptor protein. To establish that DC maturation was responsible for the adjuvant role of alphaGalCer, mice were given alphaGalCer together with soluble or cell-associated ovalbumin antigen. Th1 type CD4+ and CD8+ T cell responses developed, and the mice became resistant to challenge with ovalbumin-expressing tumor. DCs from mice given ovalbumin plus adjuvant, but not the non-DCs, stimulated ovalbumin-specific proliferative responses and importantly, induced antigen-specific, IFN-gamma producing, CD4+ and CD8+ T cells upon transfer into naive animals. In the latter instance, immune priming did not require further exposure to ovalbumin, alphaGalCer, NKT, or NK cells. Therefore a single dose of alphaGalCer i.v. rapidly stimulates the full maturation of DCs in situ, and this accounts for the induction of combined Th1 CD4+ and CD8+ T cell immunity to a coadministered protein.     

CD8(+) T cells mediate CD40-independent maturation of dendritic cells in vivo.

Induction of cytotoxic T lymphocyte (CTL) responses against minor histocompatibility antigens is dependent upon the presence of T cell help and requires the interaction of CD40 on dendritic cells (DCs) with CD40 ligand on activated T helper cells (Th). This study demonstrates that CD40 is neither involved in Th-dependent nor Th-independent antiviral CTL responses. Moreover, the data show that DC maturation occurs in vivo after viral infection in the absence of CD40 and Th. This maturation did not require viral infection of DCs but was mediated by peptide-specific CD8(+) T cells. Surprisingly, naive CD8(+) T cells were able to trigger DC maturation within 24 h after activation in vivo and in vitro. Moreover, peptide-activated CD8(+) T cells were able to induce maturation in trans, as DCs that failed to present the relevant antigen in vivo also underwent maturation. Upon isolation, the in vivo-stimulated DCs were able to convert a classically Th-dependent CTL response (anti-HY) into a Th-independent response in vitro. Thus, antiviral CD8(+) T cells are sufficient for the maturation of DCs in the absence of CD40.     

TSLP-activated dendritic cells induce an inflammatory T helper type 2 cell response through OX40 ligand

We recently showed that dendritic cells (DCs) activated by thymic stromal lymphopoietin (TSLP) prime naive CD4(+) T cells to differentiate into T helper type 2 (Th2) cells that produced high amounts of tumor necrosis factor-alpha (TNF-alpha), but no interleukin (IL)-10. Here we report that TSLP induced human DCs to express OX40 ligand (OX40L) but not IL-12. TSLP-induced OX40L on DCs was required for triggering naive CD4(+) T cells to produce IL-4, -5, and -13. We further revealed the following three novel functional properties of OX40L: (a) OX40L selectively promoted TNF-alpha, but inhibited IL-10 production in developing Th2 cells; (b) OX40L lost the ability to polarize Th2 cells in the presence of IL-12; and (c) OX40L exacerbated IL-12-induced Th1 cell inflammation by promoting TNF-alpha, while inhibiting IL-10. We conclude that OX40L on TSLP-activated DCs triggers Th2 cell polarization in the absence of IL-12, and propose that OX40L can switch IL-10-producing regulatory Th cell responses into TNF-alpha-producing inflammatory Th cell responses.     

Blocking the CD40L-CD40 interaction in vivo specifically prevents the priming of T helper 1 cells through the inhibition of interleukin 12 secretion

The recent finding that CD40L on activated T cells induces interleukin (IL) 12 secretion in human peripheral blood monocytes in vitro suggests that the CD40L-CD40 interaction may be of importance in the priming of T helper (Th) 1-type T cells. We therefore investigated the in vivo relevance of this interaction in an experimental model for a Th1- mediated disease, the hapten reagent (2,4,6-trinitrobenzene sulfonic acid [TNBS])-induced colitis. The administration of anti-gp39 (CD40L) antibodies during the induction phase of the Th1 response prevented interferon gamma production by lamina propria CD4+ T cells and also clinical and histological evidence of disease. In contrast, the secretion of IL-4, a Th2-type cytokine, was increased after anti-gp39 treatment. In further studies we showed that the prevention of disease activity was caused by an inhibition of IL-12 secretion, as demonstrated by immunohistochemistry. In addition, the injection of recombinant IL-12 p70 heterodimer into TNBS + anti-gp39-treated mice reversed the effect of anti-gp39 and resulted in severe disease activity. When anti-gp39 was given after the disease was established, no effect on the disease activity was observed. In conclusion, we demonstrated that the CD40L-CD40 interaction is crucial for the in vivo priming of Th1 T cells via the stimulation of IL-12 secretion by antigen-presenting cells (APC).     

CD8alpha- CD11b+ dendritic cells present exogenous virus-like particles to CD8+ T cells and subsequently express CD8alpha and CD205 molecules

Recombinant porcine parvovirus virus-like particles (PPV-VLPs) are particulate exogenous antigens that induce a strong, specific cytotoxic T lymphocyte (CTL) response in the absence of adjuvant. In the present report, we demonstrate in vivo that dendritic cells (DCs) present PPV-VLPs to CD8+ T cells after intracellular processing. PPV-VLPs are captured by DCs with a high efficacy, which results in the delivery of these exogenous antigens to 50% of the whole spleen DC population. In vivo, a few hours after injection, PPV-VLPs are presented exclusively to CD8+ T cells by CD8alpha- DCs, whereas 15 hours later they are presented mainly by CD8alpha+ DCs. After PPV-VLPs processing, a fraction of CD11b+ DCs undergo phenotypic changes, i.e., the up-regulation of CD8alpha and CD205 and the loss of CD4 molecules on their surface. The failure to detect mRNA coding for CD8alpha in CD11b+ DCs suggests that CD8alpha expression by these cells is not due to de novo synthesis. In recombination-activating gene knockout mice (Rag-/-), CD11b+ DCs did not express CD8alpha and PPV-VLPs presentation by CD8alpha+ DCs was severely diminished. These results indicate that both CD8alpha- and CD8alpha+ DCs play an important role in the induction of CTL responses by exogenous antigens, such as VLP.     

FBL-reactive CD8+ cytotoxic and CD4+ helper T lymphocytes recognize distinct Friend murine leukemia virus-encoded antigens

Immunization of C57BL/6 (B6) mice with FBL, a Friend murine leukemia virus (F-MuLV), induces both tumor-specific cytolytic CD8+ (CTL) and lymphokine-producing CD4+ Th that are effective in adoptive therapy of B6 mice bearing disseminated FBL leukemia. The current study evaluated the F-MuLV antigenic determinants expressed on FBL that are recognized by FBL-reactive CD8+ and CD4+ T cells. To identify the specificity of the FBL-reactive CD8+ CTL, Fisher rat embryo fibroblast (FRE) cells transfected with plasmids encoding F-MuLV gag or envelope (env) gene products plus the class I-restricting element Db were utilized. FBL-reactive CTL recognized FRE target cells transfected with the F-MuLV gag-encoded gene products, but failed to recognize targets expressing F-MuLV env. Attempts to generate env-specific CD8+ CTL by immunization with a recombinant vaccinia virus containing an inserted F-MuLV env gene were unsuccessful, despite the generation of a cytolytic response to vaccinia epitopes, implying that B6 mice fail to generate CD8+ CTL to env determinants. By contrast, CD4+ Th clones recognized FRE target cells transfected with env and not gag genes, and immunization with the recombinant vaccinia virus induced an env-specific CD4+ T cell response. These data show that in a Friend retrovirus-induced tumor model in which tumor rejection can be mediated by either CTL or Th, antigens derived from discrete retroviral proteins are predominantly responsible for activation of each T cell subset.     

Induction of anti-allo-class I H-2 tolerance by inactivation of CD8+ helper T cells, and reversal of tolerance through introduction of third-party helper T cells

The intravenous sensitization of C57BL/6 (B6) mice with class I H-2-disparate B6-C-H-2bm1 (bm1) spleen cells resulted in the abrogation of CD8+ T cell-mediated anti-bm1 (proliferative and interleukin 2-producing) T helper (Th) cell activities. In vitro stimulation of lymphoid cells from these mice with bm1 cells, however, generated a reduced, but appreciable, anti-bm1 cytotoxic T lymphocyte (CTL) response. Moreover, the anti-bm1 CTL response, upon stimulation with [bm1 x B6-C-H-2bm12 (bm12)]F1 spleen cells, was enhanced when compared with the response induced upon stimulation with bm1 cells. These in vitro results were reflected on in vivo graft rejection responses; bm1 skin grafts engrafted in the bm1-presensitized B6 mice exhibited prolonged survival, whereas (bm1 x bm12)F1 grafts placed collateral to bm1 grafts (dual engrafted mice) inhibited the tolerance to bm1. In the B6 mice 1-2 d after rejecting the bm1 grafts, anti-bm1 Th activities remained marginal, whereas potent anti-bm1 CTL responses were found to be generated from their spleen cells. Administration in vivo of anti-CD4 antibody into bm1-presensitized, dual graft-engrafted mice prolonged bm1 graft survival and interfered with enhanced induction of anti-bm1 CTL activity. These results indicate that anti-class I alloantigen (bm1) tolerance as induced by intravenous presensitization with the relevant antigens is not ascribed to the elimination of CD8+ CTL precursors, but to the specific inactivation of CD8+ Th cells, whose function can be bypassed by activating third-party Th cells.     

Generation of human CD8 T regulatory cells by CD40 ligand-activated plasmacytoid dendritic cells

Although CD8 T cell-mediated immunosuppression has been a well-known phenomenon during the last three decades, the nature of primary CD8 T suppressor cells and the mechanism underlying their generation remain enigmatic. We demonstrated that naive CD8 T cells primed with allogeneic CD40 ligand-activated plasmacytoid dendritic cells (DC)2 differentiated into CD8 T cells that displayed poor secondary proliferative and cytolytic responses. By contrast, naive CD8 T cells primed with allogeneic CD40 ligand-activated monocyte-derived DCs (DC1) differentiated into CD8 T cells, which proliferated to secondary stimulation and killed allogeneic target cells. Unlike DC1-primed CD8 T cells that produced large amounts of interferon (IFN)-gamma upon restimulation, DC2-primed CD8 T cells produced significant amounts of interleukin (IL)-10, low IFN-gamma, and no IL-4, IL-5, nor transforming growth factor (TGF)-beta. The addition of anti-IL-10-neutralizing monoclonal antibodies during DC2 and CD8 T cell coculture, completely blocked the generation of IL-10-producing anergic CD8 T cells. IL-10-producing CD8 T cells strongly inhibit the allospecific proliferation of naive CD8 T cells to monocytes, and mature and immature DCs. This inhibition was mediated by IL-10, but not by TGF-beta. IL-10-producing CD8 T cells could inhibit the bystander proliferation of naive CD8 T cells, provided that they were restimulated nearby to produce IL-10. IL-10-producing CD8 T cells could not inhibit the proliferation of DC1-preactivated effector T cells. This study demonstrates that IL-10-producing CD8 T cells are regulatory T cells, which provides a cellular basis for the phenomenon of CD8 T cell-mediated immunosuppression and suggests a role for plasmacytoid DC2 in immunological tolerance.     

CD4+ T cell polarization in mice is modulated by strain-specific major histocompatibility complex-independent differences within dendritic cells

Resistance and susceptibility to Leishmania major in mice are determined by multiple genes and correlate with the preferential development of Th1 and Th2 responses, respectively. Here, we found that CD11b+ dendritic cells (DCs) prime parasite-specific CD4+ T cells in both susceptible BALB/c (H2-d) and resistant B10.D2 (H2-d) mice. However, BALB/c and B10.D2 DCs from L. major-infected mice differ in their ability to polarize naive T cells into Th1 or Th2 effector cells. This difference is cell-intrinsic, is not restricted to H2-d mice, and is observed with both parasite-specific and allospecific CD4+ T cells. Thus, strain-specific differences within CD11b+ DCs influence the ability of inbred mice to mount polarized CD4+ T cell responses.     

IL-18-induced CD83+CCR7+ NK helper cells

In addition to their cytotoxic activities, natural killer (NK) cells can have immunoregulatory functions. We describe a distinct "helper" differentiation pathway of human CD56+CD3- NK cells into CD56+/CD83+/CCR7+/CD25+ cells that display high migratory responsiveness to lymph node (LN)-associated chemokines, high ability to produce interferon-gamma upon exposure to dendritic cell (DC)- or T helper (Th) cell-related signals, and pronounced abilities to promote interleukin (IL)-12p70 production in DCs and the development of Th1 responses. This helper pathway of NK cell differentiation, which is not associated with any enhancement of cytolytic activity, is induced by IL-18, but not other NK cell-activating factors. It is blocked by prostaglandin (PG)E2, a factor that induces a similar CD83+/CCR7+/CD25+ LN-homing phenotype in maturing DCs. The current data demonstrate independent regulation of the "helper" versus "effector" pathways of NK cell differentiation and novel mechanisms of immunoregulation by IL-18 and PGE2.     

Polarized type-1 dendritic cells (DC1) producing high levels of IL-12 family members rescue patient TH1-type antimelanoma CD4+ T cell responses in vitro

Deficiencies in TH1-type immunity in patients with cancer may facilitate tumor progression and limit the effectiveness of current immunotherapy approaches. We hypothesized that Type-1 polarized dendritic cells (DC1) might be able to recondition patient antitumor CD4+ T cell responses toward the TH1-type in vitro. Although DC1 have been previously demonstrated to prime TH1 responses from naive CD4+ T cells, their impact on antigen-experienced TH responses remains unknown. We confirmed our own earlier observations that patient CD4+ T cell reactivity against melanoma-associated antigens (MAA) was weaker and less Type-1-polarized than their corresponding antiviral responses. Stimulation of patient CD4 T cells with peptide-pulsed DC1 (producing multiple IL-12 family member cytokines, including IL-12p70, IL-23, and IL-27) promoted robust TH1-type, epitope-specific T cell responses. Addition of exogenous IL-12 family member cytokines alone, or in combination, to nonpolarized DC was insufficient to equate to the benefits associated with DC1-based stimulation; however, IL-27 and IL-12p70 blockade neutralized the ability of DC1 cells to enhance TH1-type antitumor immunity in vitro. Notably, DC1-based stimulation seemed capable of "revitalizing" defective TH1-type responses within the CD45RO+ subset of antigen-experienced CD4+ T cells in melanoma patients. In addition to promoting elevated levels of IFN-gamma from responder CD4+ T cells, DC1-based stimulation also led to increased levels of IL-12Rbeta2 and t-bet expression by TH cells. These results suggest that preexisting CD4+ T cell immunity to cancer is not relegated to Type-1 insufficiency and may be corrected via the application of DC1-based vaccination protocols.     

Modulation of cellular responses by plasmid CD40L: CD40L plasmid vectors enhance antigen-specific helper T cell type 1 CD4+ T cell-mediated protective immunity against herpes simplex virus type 2 in vivo

Engineering gene therapy vectors to modulate the immune response is an important goal. In this regard, costimulation of T cells is a critical determinant in immune activation. The costimulatory molecule CD40, expressed on antigen-presenting cells, is thought to interact with CD40 ligand (CD40L) expressed on activated CD4(+) or CD8(+) T cells to further drive interleukin-2 receptor (IL-2R) expression and antigen-specific T cell expansion necessary for both class II and class I responses. To compare the specific roles of these two costimulatory molecules in immune induction in a herpes simplex virus (HSV) model, we constructed plasmid DNAs expressing CD40 and CD40L, coimmunized these molecules with a gD plasmid vaccine, and then analyzed immune modulatory effects as well as protection against lethal HSV-2 challenge. We observed that gD-specific IgG production was unaffected by these molecules. However, a higher production of IgG2a isotype was induced by CD40L coinjection, suggesting that CD40L drives immune responses towards a helper T cell type 1 (Th1) phenotype. CD40L also enhanced Th cell proliferative responses and production of Th1-type cytokines (IL-2 and IFN-gamma) and beta-chemokines (RANTES and MIP-1alpha) from splenocytes. In contrast, CD40 showed slightly increasing effects on T cell proliferation responses and cytokine and chemokine production. When animals were challenged with a lethal dose of HSV-2, CD40L-coimmunized animals exhibited a significantly enhanced survival rate, as compared with CD40 coinjection or gD DNA vaccine alone. This enhanced protection appears to be mediated by Th1-type CD4(+) T cells, as determined by in vitro and in vivo T cell subset deletion. CD40L also promoted migration of CD4(+) T cells into the muscle sites. These studies demonstrate that CD40L can play an important role in protective antigen-specific immunity in a gene-based model system through increased expansion of the CD4(+) Th1 T cell subset in vivo.     

MHC class II expression restricted to CD8alpha+ and CD11b+ dendritic cells is sufficient for control of Leishmania major

Control of the intracellular protozoan, Leishmania major, requires major histocompatibility complex class II (MHC II)-dependent antigen presentation and CD4+ T cell T helper cell 1 (Th1) differentiation. MHC II-positive macrophages are a primary target of infection and a crucial effector cell controlling parasite growth, yet their function as antigen-presenting cells remains controversial. Similarly, infected Langerhans cells (LCs) can prime interferon (IFN)gamma-producing Th1 CD4+ T cells, but whether they are required for Th1 responses is unknown. We explored the antigen-presenting cell requirement during primary L. major infection using a mouse model in which MHC II, I-Abeta(b), expression is restricted to CD11b+ and CD8alpha+ dendritic cells (DCs). Importantly, B cells, macrophages, and LCs are all MHC II-negative in these mice. We demonstrate that antigen presentation by these DC subsets is sufficient to control a subcutaneous L. major infection. CD4+ T cells undergo complete Th1 differentiation with parasite-specific secretion of IFNgamma. Macrophages produce inducible nitric oxide synthase, accumulate at infected sites, and control parasite numbers in the absence of MHC II expression. Therefore, CD11b+ and CD8alpha+ DCs are not only key initiators of the primary response but also provide all the necessary cognate interactions for CD4+ T cell Th1 effectors to control this protozoan infection.     

Functional specialization of gut CD103+ dendritic cells in the regulation of tissue-selective T cell homing

Gut-associated lymphoid tissue (GALT) dendritic cells (DCs) display a unique ability to generate CCR9+alpha4beta7+ gut-tropic CD8+ effector T cells. We demonstrate efficient induction of CCR9 and alpha4beta7 on CD8+ T cells in mesenteric lymph nodes (MLNs) after oral but not intraperitoneal (i.p.) antigen administration indicating differential targeting of DCs via the oral route. In vitro, lamina propria (LP)-derived DCs were more potent than MLN or Peyer's patch DCs in their ability to generate CCR9+alpha4beta7+ CD8+ T cells. The integrin alpha chain CD103 (alphaE) was expressed on almost all LP DCs, a subset of MLN DCs, but on few splenic DCs. CD103+ MLN DCs were reduced in number in CCR7-/- mice and, although CD8+ T cells proliferated in the MLNs of CCR7-/- mice after i.p. but not oral antigen administration, they failed to express CCR9 and had reduced levels of alpha4beta7. Strikingly, although CD103+ and CD103- MLN DCs were equally potent at inducing CD8+ T cell proliferation and IFN-gamma production, only CD103+ DCs were capable of generating gut-tropic CD8+ effector T cells in vitro. Collectively, these results demonstrate a unique function for LP-derived CD103+ MLN DCs in the generation of gut-tropic effector T cells.     

A subset of dendritic cells induces CD4+ T cells to produce IFN-gamma by an IL-12-independent but CD70-dependent mechanism in vivo

Interferon (IFN)-gamma, a cytokine critical for resistance to infection and tumors, is produced by CD4(+) helper T lymphocytes after stimulation by cultured dendritic cells (DCs) that secrete a cofactor, interleukin (IL)-12. We have identified a major IL-12-independent pathway whereby DCs induce IFN-gamma-secreting T helper (Th)1 CD4(+) T cells in vivo. This pathway requires the membrane-associated tumor necrosis family member CD70 and was identified by targeting the LACK antigen from Leishmania major within an antibody to CD205 (DEC-205), an uptake receptor on a subset of DCs. Another major DC subset, targeted with 33D1 anti-DCIR2 antibody, also induced IFN-gamma in vivo but required IL-12, not CD70. Isolated CD205(+) DCs expressed cell surface CD70 when presenting antigen to T cell receptor transgenic T cells, and this distinction was independent of maturation stimuli. CD70 was also essential for CD205(+) DC function in vivo. Detection of the IL-12-independent IFN-gamma pathway was obscured with nontargeted LACK, which was presented by both DC subsets. This in situ analysis points to CD70 as a decision maker for Th1 differentiation by CD205(+) DCs, even in Th2-prone BALB/c animals and potentially in vaccine design. The results indicate that two DC subsets have innate propensities to differentially affect the Th1/Th2 balance in vivo and by distinct mechanisms.     

Interleukin-12 primes human CD4 and CD8 T cell clones for high production of both interferon-gamma and interleukin-10

Interleukin-12 (IL-12) induces differentiation of T helper 1 (Th1) cells, primarily through its ability to prime T cells for high interferon-gamma (IFN-gamma) production. We now report that the presence of IL-12 during the first several days of in vitro clonal expansion in limiting dilution cultures of polyclonally stimulated human peripheral blood CD4+ and CD8+ T cells also induces stable priming for high IL-10 production. This effect was demonstrated with T cells from both healthy donors and HIV+ patients. Priming for IL-4 production, which requires IL-4, was maximum in cultures containing both IL-12 and IL-4. IL-4 modestly inhibited the IL-12-induced priming for IFN-gamma, but almost completely suppressed the priming for IL-10 production. A proportion of the clones generated from memory CD45RO+ cells, but not those generated from naive CD45RO- CD4+ T cells, produced some combinations of IFN-gamma, IL-10, and IL-4 even in the absence of IL-12 and IL-4, suggesting in vivo cytokine priming; virtually all CD4+ clones generated from either CD45RO(-) or (+) cells, however, produced high levels of both IFN-gamma and IL-10 when IL-12 was present during expansion. These results indicate that each Th1-type (IFN-gamma) and Th2-type (IL-4 and IL-10) cytokine gene is independently regulated in human T cells and that the dichotomy between T cells with the cytokine production pattern of Th1 and Th2 cells is not due to a direct differentiation-inducing effect of immunoregulatory cytokines, but rather to secondary selective mechanisms. Particular combinations of cytokines induce a predominant generation of T cell clones with anomalous patterns of cytokine production (e.g., IFN-gamma and IL-4 or IFN-gamma and IL-10) that can also be found in a proportion of fresh peripheral blood T cells with "memory" phenotype or clones generated from them and that may identify novel Th subsets with immunoregulatory functions.     

Dendritic Cells Loaded With mRNA Encoding Full-length Tumor Antigens Prime CD4+ and CD8+ T Cells in Melanoma Patients

It is generally thought that dendritic cells (DCs) loaded with full-length tumor antigen could improve immunotherapy by stimulating broad T-cell responses and by allowing treatment irrespective of the patient''s human leukocyte antigen (HLA) type. To investigate this, we determined the specificity of T cells from melanoma patients treated with DCs loaded with mRNA encoding a full-length tumor antigen fused to a signal peptide and an HLA class II sorting signal, allowing presentation in HLA class I and II. In delayed-type hypersensitive (DTH)-biopsies and blood, we found functional CD8⁺ and CD4⁺ T cells recognizing novel treatment-antigen-derived epitopes, presented by several HLA types. Additionally, we identified a CD8⁺ response specific for the signal peptide incorporated to elicit presentation by HLA class II and a CD4⁺ response specific for the fusion region of the signal peptide and one of the antigens. This demonstrates that the fusion proteins contain newly created immunogenic sequences and provides evidence that ex vivo-generated mRNA-modified DCs can induce effector CD8⁺ and CD4⁺ T cells from the naive T-cell repertoire of melanoma patients. Thus, this work provides definitive proof that DCs presenting the full antigenic spectrum of tumor antigens can induce T cells specific for novel epitopes and can be administered to patients irrespective of their HLA type.