No abstract

Active T cells release bioactive exosomes (EXOs). However, its potential modulation in immune responses is elusive. In this study, we in vitro generated active OVA-specific CD8+ T cells by cultivation of OVA-pulsed dendritic cells (DCOVA) with naive CD8+ T cells derived from OVA-specific TCR transgenic OTI mice and purified EXOs from CD8+ T cell culture supernatant by differential ultracentrifugation. We then investigated the suppressive effect of T cell EXOs on DCOVA-mediated CD8+ CTL responses and antitumor immunity. We found that DCOVA uptake OTI T cell EXOs expressing OVA-specific TCRs and Fas ligand via peptide/MHC Ag I–TCR and CD54–LFA-1 interactions leading to downregulation of peptide/MHC Ag I expression and induction of apoptosis of DCOVA via Fas/Fas ligand pathway. We demonstrated that OVA-specific OTI T cell EXOs, but not lymphocytic choriomeningitis virus-specific TCR transgenic mouse CD8+ T cell EXOs, can inhibit DCOVA-stimulated CD8+ CTL responses and antitumor immunity against OVA-expressing B16 melanoma. In addition, these T cell EXOs can also inhibit DCOVA-mediated CD8+ CTL-induced diabetes in transgenic rat insulin promoter-mOVA mice. Interestingly, the anti–LFA-1 Ab treatment significantly reduces T cell EXO-induced inhibition of CD8+ CTL responses in both antitumor immunity and autoimmunity. EXOs released from T cell hybridoma RF3370 cells expressing OTI CD8+ TCRs have a similar inhibitory effect as T cell EXOs in DCOVA-stimulated CTL responses and antitumor immunity. Therefore, our data indicate that Ag-specific CD8+ T cells can modulate immune responses via T cell-released EXOs, and T cell EXOs may be useful for treatment of autoimmune diseases.

CD4(+) Th17 cells induce antitumor immunity leading to the eradication of established tumors. However, the mechanism of antitumour immunity and CTL activation by Th17 cells and the distinct role of Th17 and Th17-activated CTLs in antitumor immunity are still elusive. In this study, we generated ovalbumin (OVA)-specific Th17 cells by cultivating OVA-pulsed dendritic cells with CD4(+) T cells derived from transgenic OTII mice in the presence of IL-6, IL-23, TGF-β, and anti-IFN-γ antibody. We demonstrated that Th17 cells acquired major histocompatibility complex/peptide (pMHC)-I and expressed RORγt, IL-17, and IL-2. Th17 cells did not have any direct in vitro tumor cell-killing activity. However, Th17 cells were able to stimulate CD8(+) CTL responses via IL-2 and pMHC I, but not IL-17 signaling, which play a major role in Th17-induced preventive immunity against OVA-expressing B16 melanoma. Th17 cells stimulated the expression of CCL2 and CCL20 in lung tumor microenvironments promoting the recruitment of various inflammatory leukocytes (DCs, CD4(+), and CD8(+) T cells) stimulating more pronounced therapeutic immunity for early-stage (5-day lung metastases or 3 mm, s.c.) tumor than for well-established (6 mm, s.c.) tumor. The therapeutic effect of Th17 cells is associated with IL-17 and is mediated by Th17-stimulated CD8(+) CTLs and other inflammatory leukocytes recruited into B16 melanoma via Th17-stimulated CCL20 chemoattraction. Taken together, our data elucidate a distinct role of Th17 and Th17-stimulated CD8(+) CTLs in the induction of preventive and therapeutic antitumor immunity, which may greatly impact the development of Th17-based cancer immunotherapy.

CD4(+) T cell help contributes critically to DC-induced CD8(+) CTL immunity. However, precisely how these three cell populations interact and how CD4(+) T cell signals are delivered to CD8(+) T cells in vivo have been unclear. In this study, we developed a novel, two-step approach, wherein CD4(+) T cells and antigen-presenting DCs productively engaged one another in vivo in the absence of cognate CD8(+) T cells, after which, we selectively depleted the previously engaged CD4(+) T cells or DCs before allowing interactions of either population alone with naïve CD8(+) T cells. This protocol thus allows us to clearly document the importance of CD4(+) T-licensed DCs and DC-primed CD4(+) T cells in CTL immunity. Here, we provide direct in vivo evidence that primed CD4(+) T cells or licensed DCs can stimulate CTL response and memory, independent of DC-CD4(+) T cell clusters. Our results suggest that primed CD4(+) T cells with acquired pMHC-I from DCs represent crucial "immune intermediates" for rapid induction of CTL responses and for functional memory via CD40L signaling. Importantly, intravital, two-photon microscopy elegantly provide unequivocal in vivo evidence for direct CD4-CD8(+) T cell interactions via pMHC-I engagement. This study corroborates the coexistence of direct and indirect mechanisms of T cell help for a CTL response in noninflammatory situations. These data suggest a new "dynamic model of three-cell interactions" for CTL immunity derived from stimulation by dissociated, licensed DCs, primed CD4(+) T cells, and DC-CD4(+) T cell clusters and may have significant implications for autoimmunity and vaccine design.

The Ag-specific CD4+ regulatory T (Tr) cells play an important role in immune suppression in autoimmune diseases and antitumor immunity. However, the molecular mechanism for Ag-specificity acquisition of adoptive CD4+ Tr cells is unclear. In this study, we generated IL-10- and IFN-γ-expressing type 1 CD4+ Tr (Tr1) cells by stimulation of transgenic OT II mouse-derived naive CD4+ T cells with IL-10-expressing adenovirus (AdVIL-10)-transfected and OVA-pulsed dendritic cells (DCOVA/IL-10). We demonstrated that both in vitro and in vivo DCOVA/IL-10-stimulated CD4+ Tr1 cells acquired OVA peptide MHC class (pMHC) I which targets CD4+ Tr1 cells suppressive effect via an IL-10-mediated mechanism onto CD8+ T cells, leading to an enhanced suppression of DCOVA-induced CD8+ T cell responses and antitumor immunity against OVA-expressing murine B16 melanoma cells by ≈700% relative to analogous CD4+ Tr1 cells without acquired pMHC I. Interestingly, the nonspecific CD4+25+ Tr cells can also become OVA Ag specific and more immunosuppressive in inhibition of OVA-specific CD8+ T cell responses and antitumor immunity after uptake of DCOVA-released exosomal pMHC I complexes. Taken together, the Ag-specificity acquisition of CD4+ Tr cells via acquiring DC’s pMHC I may be an important mean in augmenting CD4+ Tr cell suppression.

TLR ligands have been reported to promote DC maturation and enhance CD8+ CTL responses. We have demonstrated previously that CD4-8- DCs secreting TGF-beta stimulate CD4+ Tr1 cell responses. Here, we have assessed whether TLR4 and TLR9 signaling through LPS and CpG stimulation can convert CD4-8- DC-induced tolerance. We demonstrate that immature OVA-pulsed CD4-8- DCs cultured in medium with LPS (2 microg/ml) and CpG (5 microg/ml) for 8 h became mature DCs (DCOVA) with no TGF-beta secretion. CpG-treated, CD4-8- DCOVA-secreting IL-6/IL-15 induced IFN-gamma/IL-17-secreting/T-bet- and ROR-gammat-expressing CD4+ Th1/Th17, whereas LPS-treated CD4-8- DCOVA stimulated IFN-gamma-secreting/T-bet-expressing CD4+ Th1 responses. The former also significantly stimulated more efficient OVA-specific CD8+ T cell responses and antitumor immunity against OVA-expressing BL6-10OVA tumor cells than the latter (P<0.05). CpG-treated, CD4-8- DCOVA-stimulated CD4+ Th1/Th17 cell responses and antitumor immunity were found to be reduced by using neutralizing anti-IL-6, IL-15, and NK1.1 antibodies in wild-type C57BL/6 mice, IL-15R-/- mice for immunization, or CD4-8- (IL-6-/-) DCOVA for immunization in C57BL/6 mice. Interestingly, in vitro-generated CD4+ Th17 cells significantly enhanced LPS-treated, CD4-8- DCOVA-induced in vivo antitumor immunity via increasing CD8+ CTL responses (P<0.05), although they did not show any direct killing activity against tumor cells in vitro. In addition, prolonged 48 h CpG-treated CD4-8- DCOVA dramatically diminished its cytokine secretion, stimulatory effect, and antitumor immunity. Taken together, our data demonstrate an effect of conversion of tolerogenic DCs into immunogenic ones capable of stimulating antitumor immunity via activating CD4+ Th1/Th17 and NK cell responses by optimal CpG signaling, which may advance current understanding of the importance of TLR9 signaling in a DC-based cancer vaccine.

Both CD4+ Th17-cells and CD8+ cytotoxic T lymphocytes (CTLs) are involved in type 1 diabetes and experimental autoimmune encephalomyelitis (EAE). However, their relationship in pathogenesis of these autoimmune diseases is still elusive. We generated ovalbumin (OVA)- or myelin oligodendrocyte glycoprotein (MOG)- specific Th17 cells expressing RORγt and IL-17 by in vitro co-culturing OVA-pulsed and MOG35-55 peptide-pulsed dendritic cells (DCOVA and DCMOG) with CD4+ T cells derived from transgenic OTII and MOG-T cell receptor mice, respectively. We found that these Th17 cells when transferred into C57BL/6 mice stimulated OVA- and MOG-specific CTL responses, respectively. To assess the above question, we adoptively transferred OVA-specific Th17 cells into transgenic rat insulin promoter (RIP)-mOVA mice or RIP-mOVA mice treated with anti-CD8 antibody to deplete Th17-stimulated CD8+ T cells. We demonstrated that OVA-specific Th17-stimulated CTLs, but not Th17 cells themselves, induced diabetes in RIP-mOVA. We also transferred MOG-specific Th17 cells into C57BL/6 mice and H-2Kb−/− mice lacking of the ability to generate Th17-stimulated CTLs. We further found that MOG-specific Th17 cells, but not Th17-activated CTLs induced EAE in C57BL/6 mice. Taken together, our data indicate a distinct role of Th17 cells and Th17-stimulated CTLs in the pathogenesis of TID and EAE, which may have great impact on the overall understanding of Th17 cells in the pathogenesis of autoimmune diseases.

Gene therapy has been considered as the most ideal medical intervention for genetic diseases because it is intended to target the cause of diseases instead of disease symptoms. Availability of techniques for identification of genetic mutations and for in vitro manipulation of genes makes it practical and attractive. After the initial hype in 1990s and later disappointments in clinical trials for more than a decade, light has finally come into the tunnel in recent years, especially in the field of eye gene therapy where it has taken big strides. Clinical trials in gene therapy for retinal degenerative diseases such as Leber's congenital amaurosis (LCA) and choroideremia demonstrated clear therapeutic efficacies without apparent side effects. Although these successful examples are still rare and sporadic in the field, they provide the proof of concept for harnessing the power of gene therapy to treat genetic diseases and to modernize our medication. In addition, those success stories illuminate the path for the development of gene therapy treating other genetic diseases. Because of the differences in target organs and cells, distinct barriers to gene delivery exist in gene therapy for each genetic disease. It is not feasible for authors to review the current development in the entire field. Thus, in this article, we will focus on what we can learn from the current success in gene therapy for retinal degenerative diseases to speed up the gene therapy development for lung diseases, such as cystic fibrosis.