IL-12 in conjunction with dendritic cells enhances antiviral CD8+ CTL responses in vitro.

CD8+ cytolytic T lymphocytes (CTLs) are important mediators for resistance to infections and malignant diseases. IL-12 enhances proliferative and cytolytic responses by killer cells, but its function in the generation of human antiviral CD8+ T cell responses has not been defined. We therefore evaluated the role of IL-12 in the generation of CTLs to influenza-infected dendritic cells. IL-12 was not detectable in supernatants of infected-dendritic cells, or during CTL generation. Furthermore, anti-IL-12 antibody did not block CTL generation. However, exogenous IL-12 (30-300 pg/ml) enhanced CD8+ T cell proliferative and cytolytic responses. The effect was greatest in individuals with weak reactivity to influenza virus or at antigen-presenting cell (APC):T cell ratios of 1:100 or less. IL-12 augmented interferon-gamma production during CTL generation. The CTL enhancing effects of the cytokine, however, could not be blocked by neutralizing anti-interferon-gamma antibody. Together with IL-12, antigen-pulsed dendritic cells may be a useful approach for boosting CTL responses against infectious agents and malignancies.     

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.     

Regulatory CD4+CD25+ T cells restrict memory CD8+ T cell responses

CD4+ T cell help is important for the generation of CD8+ T cell responses. We used depleting anti-CD4 mAb to analyze the role of CD4+ T cells for memory CD8+ T cell responses after secondary infection of mice with the intracellular bacterium Listeria monocytogenes, or after boost immunization by specific peptide or DNA vaccination. Surprisingly, anti-CD4 mAb treatment during secondary CD8+ T cell responses markedly enlarged the population size of antigen-specific CD8+ T cells. After boost immunization with peptide or DNA, this effect was particularly profound, and antigen-specific CD8+ T cell populations were enlarged at least 10-fold. In terms of cytokine production and cytotoxicity, the enlarged CD8+ T cell population consisted of functional effector T cells. In depletion and transfer experiments, the suppressive function could be ascribed to CD4+CD25+ T cells. Our results demonstrate that CD4+ T cells control the CD8+ T cell response in two directions. Initially, they promote the generation of a CD8+ T cell responses and later they restrain the strength of the CD8+ T cell memory response. Down-modulation of CD8+ T cell responses during infection could prevent harmful consequences after eradication of the pathogen.     

CD4+CD25+ T cells regulate virus-specific primary and memory CD8+ T cell responses

Naturally occurring CD4+CD25+ regulatory T cells appear important to prevent activation of autoreactive T cells. This article demonstrates that the magnitude of a CD8+ T cell-mediated immune response to an acute viral infection is also subject to control by CD4+CD25+ T regulatory cells (Treg). Accordingly, if natural Treg were depleted with specific anti-CD25 antibody before infection with HSV, the resultant CD8+ T cell response to the immunodominant peptide SSIEFARL was significantly enhanced. This was shown by several in vitro measures of CD8+ T cell reactivity and by assays that directly determine CD8+ T cell function, such as proliferation and cytotoxicity in vivo. The enhanced responsiveness in CD25-depleted animals was between three- and fourfold with the effect evident both in the acute and memory phases of the immune response. Surprisingly, HSV infection resulted in enhanced Treg function with such cells able to suppress CD8+ T cell responses to both viral and unrelated antigens. Our results are discussed both in term of how viral infection might temporarily diminish immunity to other infectious agents and their application to vaccines. Thus, controlling suppressor effects at the time of vaccination could result in more effective immunity.     

Complementary dendritic cell-activating function of CD8+ and CD4+ T cells: helper role of CD8+ T cells in the development of T helper type 1 responses

Dendritic cells (DCs) activated by CD40L-expressing CD4+ T cells act as mediators of "T helper (Th)" signals for CD8+ T lymphocytes, inducing their cytotoxic function and supporting their long-term activity. Here, we show that the optimal activation of DCs, their ability to produce high levels of bioactive interleukin (IL)-12p70 and to induce Th1-type CD4+ T cells, is supported by the complementary DC-activating signals from both CD4+ and CD8+ T cells. Cord blood- or peripheral blood-isolated naive CD8+ T cells do not express CD40L, but, in contrast to naive CD4+ T cells, they are efficient producers of IFN-gamma at the earliest stages of the interaction with DCs. Naive CD8+ T cells cooperate with CD40L-expressing naive CD4+ T cells in the induction of IL-12p70 in DCs, promoting the development of primary Th1-type CD4+ T cell responses. Moreover, the recognition of major histocompatibility complex class I-presented epitopes by antigen-specific CD8+ T cells results in the TNF-alpha- and IFN-gamma-dependent increase in the activation level of DCs and in the induction of type-1 polarized mature DCs capable of producing high levels of IL-12p70 upon a subsequent CD40 ligation. The ability of class I-restricted CD8+ T cells to coactivate and polarize DCs may support the induction of Th1-type responses against class I-presented epitopes of intracellular pathogens and contact allergens, and may have therapeutical implications in cancer and chronic infections.     

Tumor cells convert immature myeloid dendritic cells into TGF-beta-secreting cells inducing CD4+CD25+ regulatory T cell proliferation

The mechanisms through which regulatory T cells accumulate in lymphoid organs of tumor-bearing hosts remain elusive. Our experiments indicate that the accumulation of CD4+CD25+ regulatory T cells (T reg cells) expressing FoxP3 and exhibiting immunosuppressive function originates from the proliferation of naturally occurring CD25+ T cells and requires signaling through transforming growth factor (TGF)-beta receptor II. During tumor progression, a subset of dendritic cells (DCs) exhibiting a myeloid immature phenotype is recruited to draining lymph nodes. This DC subset selectively promotes the proliferation of T reg cells in a TGF-beta-dependent manner in mice and rats. Tumor cells are necessary and sufficient to convert DCs into regulatory cells that secrete bioactive TGF-beta and stimulate T reg cell proliferation. In conclusion, tumor expansion can stimulate T reg cells via a specific DC subset.     

T helper cell 1-type CD4+ T cells, but not B cells, mediate colitis in interleukin 10-deficient mice

Mice rendered deficient in the production of interleukin 10 (IL-10-/-) develop a chronic inflammatory bowel disease (IBD) that predominates in the colon and shares histopathological features with human IBD. Our aim was to identify which cell type(s) can mediate colitis in IL-10-/- mice. We detected an influx of immunoglobulin-positive cells into the colon and the presence of colon-reactive antibodies in the serum of IL- 10-/- mice. To assess a pathogenic role for B cells, we generated a B cell-deficient (B-/-) strain of IL-10-/- mice. B-/-IL-10-/- mice acquired a severe colitis analogous to that IL-10-/- mice, implying that B cells were not the primary mediator of IBD in this model. A series of cell transfer experiments was performed to assess a pathogenic role for T cells. When IL-10-/- T cell-enriched lamina propria lymphocytes (LPL) or intraepithelial lymphocytes (IEL) were transferred into immunodeficient recombinase-activating gene (RAG)-2-/- recipients, a mild to severe colitis developed, depending on the cell number transferred. Lymphocytes recovered from the colon of transplanted RAG-2-/- mice with colitis were predominantly alpha beta TCR+CD4+, including a large proportion of CD4+CD8 alpha + cells. These cells were also CD45RB-/low and CD44+, indicative of an activated/memory population. Individual populations of CD4+CD8 alpha-, CD4+CD8 alpha + and CD4-CD8 alpha + T cells were then isolated from the lamina propria compartment of IL-10-/- mice and transferred into RAG-2- /- recipients. Only IL-10-/- CD4-expressing LPL, including both the CD4+CD8 alpha- and CD4+CD8 alpha + populations, induced colitis in recipient mice. Interferon-gamma, but little to no IL-4, was produced by CD4+CD8 alpha- and CD4+CD8 alpha + LPL recovered from the inflamed colons of RAG-2-/- recipients implicating alpha T helper cell 1 (TH1)- mediated response. We thus conclude that colitis in IL-10-/- mice is predominantly mediated by TH1-type alpha beta TCR+ T cells expressing CD4 alone, or in combination with the CD8 alpha molecule.     

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.     

Induction of B cell apoptosis by TH0, but not TH2, CD4+ T cells.

Engagement of the T cell receptor molecules with MHC-antigen complexes presented by B cells ascertains antigen specificity in T cell-dependent help. Ligation of MHC molecules on the surface of B cells, however, has not only been implicated in antigen-specific T-B cell interaction, but has also been linked to the induction of B cell apoptosis. To examine the role of T helper cells in either induction of immunoglobulin synthesis or B cell apoptotic death, we have facilitated T cell receptor-MHC interaction through a bacterial superantigen. CD4+ T cell clones could be categorized into two clearly distinct subsets based upon their ability to promote B cell help in the presence of superantigen. One subset of T cell clones supported immunoglobulin synthesis, and thus functioned as effective helper cells. B cells interacting with the second subset of T cells did not differentiate into antibody-secreting cells, but underwent apoptosis. Both types of helper cells were able to provide contact help after anti-CD3 stimulation. Induction of apoptosis was a dominant phenomenon; the addition of the superantigen suppressed immunoglobulin production in B cells activated by anti-CD3-stimulated helper T cells, indicating that the T cells delivered an apoptotic signal to the B cell. T cell clones providing effective MHC restrictive B cell help could be distinguished from T cells facilitating B cell apoptosis based on their lymphokine secretion profile. Induction of B cell apoptosis was a feature of T cells with a TH0 lymphokine pattern. Promotion of MHC-restricted B cell help was associated with a TH2 lymphokine profile. TH1-derived cytokines alone could not substitute for apoptosis-inducing T cells.     

Myeloid and plasmacytoid dendritic cells transfer HIV-1 preferentially to antigen-specific CD4+ T cells

Dendritic cells (DCs) are essential antigen-presenting cells for the induction of T cell immunity against pathogens such as human immunodeficiency virus (HIV)-1. At the same time, HIV-1 replication is strongly enhanced in DC-T cell clusters, potentially undermining this process. We found that immature CD123(+) plasmacytoid DCs (PDCs) and CD11c(+) myeloid DCs (MDCs) were susceptible to both a CCR5- and a CXCR4-using HIV-1 isolate in vitro and were able to efficiently transfer that infection to autologous CD4(+) T cells. Soon after HIV-1 exposure, both PDCs and MDCs were able to transfer the virus to T cells in the absence of a productive infection. However, once a productive infection was established in the DCs, newly synthesized virus was predominantly spread to T cells. HIV-1 exposure of the MDCs and PDCs did not inhibit their ability to present cytomegalovirus (CMV) antigens and activate CMV-specific memory T cells. As a result, both PDCs and MDCs preferentially transmitted HIV-1 to the responding CMV antigen-specific CD4(+) T cells rather than to nonresponding T cells. This suggests that the induction of antigen-specific T cell responses by DCs, a process crucial to immune defense, can lead to preferential HIV-1 infection and the deletion of responding CD4(+) T cells.     

Targeting self- and foreign antigens to dendritic cells via DC-ASGPR generates IL-10-producing suppressive CD4+ T cells

Dendritic cells (DCs) can initiate and shape host immune responses toward either immunity or tolerance by their effects on antigen-specific CD4(+) T cells. DC-asialoglycoprotein receptor (DC-ASGPR), a lectinlike receptor, is a known scavenger receptor. Here, we report that targeting antigens to human DCs via DC-ASGPR, but not lectin-like oxidized-LDL receptor, Dectin-1, or DC-specific ICAM-3-grabbing nonintegrin favors the generation of antigen-specific suppressive CD4(+) T cells that produce interleukin 10 (IL-10). These findings apply to both self- and foreign antigens, as well as memory and naive CD4(+) T cells. The generation of such IL-10-producing CD4(+) T cells requires p38/extracellular signal-regulated kinase phosphorylation and IL-10 induction in DCs. We further demonstrate that immunization of nonhuman primates with antigens fused to anti-DC-ASGPR monoclonal antibody generates antigen-specific CD4(+) T cells that produce IL-10 in vivo. This study provides a new strategy for the establishment of antigen-specific IL-10-producing suppressive T cells in vivo by targeting whole protein antigens to DCs via DC-ASGPR.     

CD4+CD25+ T cells protect against experimentally induced asthma and alter pulmonary dendritic cell phenotype and function

The role of natural CD4+CD25+ regulatory T (T reg) cells in the control of allergic asthma remains poorly understood. We explore the impact of T reg cell depletion on the allergic response in mice susceptible (A/J) or comparatively resistant (C3H) to the development of allergen-induced airway hyperresponsiveness (AHR). In C3H mice, anti-CD25-mediated T reg cell depletion before house dust mite treatment increased several features of the allergic diathesis (AHR, eosinophilia, and IgE), which was concomitant with elevated T helper type 2 (Th2) cytokine production. In similarly T reg cell-depleted A/J mice, we observed a moderate increase in airway eosinophilia but no effects on AHR, IgE levels, or Th2 cytokine synthesis. As our experiments suggested that T reg cell depletion in C3H mice before sensitization was sufficient to enhance the allergic phenotype, we characterized dendritic cells (DCs) in T reg cell-depleted C3H mice. T reg cell-depleted mice had increased numbers of pulmonary myeloid DCs with elevated expression of major histocompatibility complex class II, CD80, and CD86. Moreover, DCs from T reg cell-depleted mice demonstrated an increased capacity to stimulate T cell proliferation and Th2 cytokine production, which was concomitant with reduced IL-12 expression. These data suggest that resistance to allergen-driven AHR is mediated in part by CD4+CD25+ T reg cell suppression of DC activation and that the absence of this regulatory pathway contributes to susceptibility.     

Imaging the single cell dynamics of CD4+ T cell activation by dendritic cells in lymph nodes

The adaptive immune response is initiated in secondary lymphoid organs by contact between antigen-bearing dendritic cells (DCs) and antigen-specific CD4+ T cells. However, there is scant information regarding the single cell dynamics of this process in vivo. Using two-photon microscopy, we imaged the real-time behavior of naive CD4+ T cells and in vivo-labeled DCs in lymph nodes during a robust T cell response. In the first 2 h after entry into lymph nodes, T cells made short-lived contacts with antigen-bearing DCs, each contact lasting an average of 11-12 min and occurring mainly on dendrites. Altered patterns of T cell motility during this early stage of antigen recognition promoted serial engagement with several adjacent DCs. Subsequently, T cell behavior progressed through additional distinct stages, including long-lived clusters, dynamic swarms, and finally autonomous migration punctuated by cell division. These observations suggest that the immunological synapse in native tissues is remarkably fluid, and that stable synapses form only at specific stages of antigen presentation to T cells. Furthermore, the serial nature of these interactions implies that T cells activate by way of multiple antigen recognition events.     

CD40L+ CD4+ memory T cells migrate in a CD62P-dependent fashion into reactive lymph nodes and license dendritic cells for T cell priming

There is growing evidence that the maturation state of dendritic cells (DCs) is a critical parameter determining the balance between tolerance and immunity. We report that mouse CD4⁺ effector memory T (T_EM) cells, but not naive or central memory T cells, constitutively expressed CD40L at levels sufficient to induce DC maturation in vitro and in vivo in the absence of antigenic stimulation. CD4⁺ T_EM cells were excluded from resting lymph nodes but migrated in a CD62P-dependent fashion into reactive lymph nodes that were induced to express CD62P, in a transient or sustained fashion, on high endothelial venules. Trafficking of CD4⁺ T_EM cells into chronic reactive lymph nodes maintained resident DCs in a mature state and promoted naive T cell responses and experimental autoimmune encephalomyelitis (EAE) to antigens administered in the absence of adjuvants. Antibodies to CD62P, which blocked CD4⁺ T_EM cell migration into reactive lymph nodes, inhibited DC maturation, T cell priming, and induction of EAE. These results show that T_EM cells can behave as endogenous adjuvants and suggest a mechanistic link between lymphocyte traffic in lymph nodes and induction of autoimmunity.     

Role of IL-2 secreted by PADRE-specific CD4+ T cells in enhancing E7-specific CD8+ T-cell immune responses

CD4(+) T helper cells are known to play an integral role in the generation of CD8(+) T-cell immune responses. We have previously shown that co-administration of DNA vaccines containing E6 or E7 protein of human papillomavirus 16 (HPV-16) combined with DNA encoding invariant (Ii) chain in which class II-associated Ii peptide (CLIP) region is replaced with the CD4(+) T helper epitope, PADRE (Pan-DR-epitope) (Ii-PADRE DNA) enhanced HPV antigen-specific CD8(+) T-cell immune responses in vaccinated mice. In the current study, we investigated the enhancement of HPV E7-specific CD8(+) T-cell immune responses by PADRE-specific CD4(+) T cells. We showed that intradermal administration of Ii-PADRE DNA at the same location as E7-expressing DNA is necessary to generate strong E7-specific CD8(+) T-cell immune responses. We also showed that PADRE-specific CD4(+) T cells generated by Ii-PADRE DNA vaccination expressed Th1 cytokine profile. Furthermore, our in vitro study demonstrated that PADRE-specific CD4(+) T cells stimulated with PADRE-loaded dendritic cells secrete IL-2 that leads to the proliferation of E7-specific CD8(+) T cells. Thus, our data suggest that activated PADRE-specific CD4(+) T helper cells may be required at the vicinity of E7-specific CD8(+) T cells where they secrete IL-2, which enhances the E7-specific CD8(+) T-cell immune responses generated by DNA vaccination.     

cDC1-derived IL-27 regulates small intestinal CD4+ T cell homeostasis in mice

The small intestinal lamina propria contains large numbers of IFNγ-producing T helper (Th1) cells that play important roles in intestinal homeostasis and host defense, but the mechanisms underlying their development remain poorly understood. Here, we demonstrate that Th1 cells accumulate in the SI-LP after weaning and are maintained there long term. While both Th17 and Th1 cell accumulation in the SI-LP was microbiota dependent, Th1 cell accumulation uniquely required IL-27 and MHCII expression by cDC1. This reflected a requirement for IL-27 signaling in the priming of Th1 cells rather than for their maintenance once in the mucosa. cDC1-derived IL-27 was essential for maintaining the Th1–Th17 balance within the SI-LP, and in its absence, remaining Th1 cells expressed enhanced levels of Th17 signature genes. In conclusion, we identify cDC1-derived IL-27 as a key regulator of SI-LP Th1–Th17 cell homeostasis.     

Interferon gamma enhances both in vitro and in vivo priming of CD4+ T cells for IL-4 production

Classical studies have demonstrated that in vitro priming of naive CD4 T cells to become T helper (Th)2 cells is strikingly dependent on interleukin (IL)-4, whereas priming for interferon (IFN)gamma production is IL-12/IFNgamma-dependent. Therefore, it was quite surprising when we noted that priming of naive C57BL/6 CD4(+) cells to become IL-4 producers was substantially inhibited by the addition of anti-IFNgamma antibodies. This was true using immobilized anti-CD3 and anti-CD28 antibodies or soluble anti-CD3/anti-CD28 and antigen-presenting cells in the presence or absence of added IL-4. Priming of CD4 T cells from IFNgamma(-/-) C57BL/6 mice with immobilized anti-CD3 and anti-CD28 resulted in limited production of IL-4, even with the addition of 1,000 U/ml of IL-4. Titrating IFNgamma into such cultures showed a striking increase in the proportion of T cells that secreted IL-4 upon challenge; this effect was completely IL-4-dependent in that it was blocked with anti-IL-4 antibody. Thus, IFNgamma plays an unanticipated but substantial role in Th2 priming, although it is an important Th1 cytokine, and under certain circumstances a Th1 inducer.