Tolerogenic CD4⁻8⁻ Dendritic Cells and their Conversion into Immunogenic Ones via TLR9 Signaling
It is clear that dendritic cells (DCs) are essential for priming of T cell responses against tumors. However, the distinct roles DC subsets play in regulation of T cell responses in vivo are largely undefined. In this study, we investigated the capacity of ovalbumin (OVA)-presenting CD4⁻8⁻, CD4⁺8⁻, or CD4⁻8⁺ DCs (OVA-pulsed DC (DCOVA)) from mouse spleen in stimulation of OVA-specific T cell responses. Our data show that each DC subset stimulated proliferation of allogeneic and autologous OVA-specific CD4⁺ and CD8⁺ T cells in vitro, but that the CD4⁻8⁻ DCs did so only weakly. Both CD4⁺8⁻ and CD4⁻8⁺ DCOVA induced strong tumor-specific CD4⁺ Th1 responses and fully protective CD8⁺ cytotoxic T lymphocyte (CTL)-mediated antitumor immunity, whereas CD4⁻8⁻ DCOVA, which were less mature and secreted substantial transforming growth factor (TGF-β ) upon coculture with T cell receptor (TCR)-transgenic OT II CD4⁺ T cells, induced the development of interleukin-10 (IL-10)-secreting CD4⁺ T regulatory 1 (Tr1) cells. Transfer of these Tr1 cells, but not T cells from cocultures of CD4⁻8⁻ DCOVA and IL-10⁻/⁻ OT II CD4⁺ T cells, into CD4⁻8⁺ DCOVA-immunized animals abrogated otherwise inevitable development of antitumor immunity. Taken together, CD4⁻8⁻ DCs stimulate development of IL-10-secreting CD4⁺ Tr1 cells that mediated immune suppression, whereas both CD4⁺8⁻ and CD4⁻8⁺ DCs effectively primed animals for protective CD8⁺ CTL-mediated antitumor immunity. Different DC subsets play distinct roles in immune responses. CD4⁻8⁻ DCs secreting TGF-β stimulate CD4⁺ regulatory T type 1 (Trl) cell responses leading to inhibition of CD8 CTL responses and antitumor immunity. In this study, we explored the potential effect of three stimuli CpG, lipopolysaccharide (LPS) and anti-CD40 antibody in conversion of CD4⁻8⁻ DC-induced tolerance. We demonstrated that when CD4⁻8⁻ DCs were isolated from overnight culture and cultured for another 8 hrs in AIM-V plus recombinant mouse granulocyte-macrophage colony-stimulating factor (rmGM-CSF) (15-20 ng/ml) and OVA (0.1 mg/ml) with CpG (5 ug/ml), LPS (2 ug/ml) and anti-CD40 antibody (10 ug/ml), their phenotype became more mature compared with the freshly isolated ones. CpG is the only agent that stimulates the DCs to secrete significant level of interleukin-6 (IL-6) and interleukin-15 (IL-15); DNA array analyses also indicate that CpG stimulates higher expression of IL-6 and IL-15 mRNA. CpG treatment most efficiently converts the tolerogenic DCs into immunogenic ones which stimulated the OTII CD4⁺ T cell to become T helper type 1 (Th1) and T helper type 17 (Th17) rather Tr1, while the other two stimulator-treated DCs could not induce Th17 response. Their vaccination also induced the strongest antitumor CTL responses and protective immunity against tumor cell challenge. When CD4⁻8⁻ DCs were isolated from IL-6 knock out (IL-6⁻/⁻) mice, CpG-treated DCOVA vaccination almost completely lost their animal protection capacity. Wild type B6 DCOVA-vaccinated IL-15 receptor knock out (IL-15R⁻/⁻) mice can only provide up to 30% protection against tumor challenge. Those results indicate that IL-6/ IL-l5-induced Th17 plays a critical role in their conversion. Taken together, our findings indicate that CpG treatment is the most efficient agent that can convert tolerogenic DCs into immunogenic ones and induce long-lasting antitumor immunity. We previously demonstrated that the nonspecific CD4⁺ T cells can acquire antigen-specific DC-released exosomes (EXO) and these CD4⁺ T cells with acquired exosomal MHC I peptide complex (pMHC I) can stimulate antigen-specific CD8⁺ CTL responses. In my project we have found that CD4⁻8⁻DCs could induce regulatory T cell type 1(Tr1) response, thus it would be very necessary to know whether regulatory T cells would change their antigen specificity if they got the membrane complex from DC through coculture or DC-derived exosome pulsing. During the beginning of my regulatory T cell project, we found that CD8⁺CD25⁺ Tr were much more easily expanded, while CD4⁺CD25⁺ Tr usually began to die just after 3 days in vitro culture and it’s very hard to get enough cells for further research. Therefore, CD8⁺CD25⁺ were used as a model Tr cells in the following project. To assess whether the nonspecific CD8⁺CD25⁺ Tr cells can acquire antigen-specificity via acquired exosomal pMHC I, we purified CD8⁺CD25⁺ Tr cells from wild-type C57BL/6 mice and OVA-pulsed DCOVA-released EXOOVA expressing pMHC I complexes. We demonstrated that the nonspecific CD8⁺CD25⁺ Tr cells expressing forkhead box P3 (Foxp3), cytotoxic T-Lymphocyte Antigen 4 (CTLA-4), glucocorticoid-induced tumor necrosis factor receptor (GITR), perforin and granzyme B inhibited in vitro T cell proliferation and in vivo OVA-specific CD4⁺ T cell-dependent and independent CD8⁺ CTL responses and antitumor immunity. CD8⁺CD25⁺ Tr cells’ suppressive effect is possibly mediated through its inhibition of DC maturation, down-regulation of secretion of Th1 polarization cytokines by DCs and its induction of T cell anergy via cell-to-cell contact. The nonspecific CD8⁺CD25⁺ Tr cells acquired antigen specificity by uptake of DCOVA-released EXOOVA expressing pMHC I and enhanced its effect on inhibition of OVA-specific CD8⁺ T cell responses and antitumor immunity by 10-folds. The principles elucidated in this study may have significant implications not only in antitumor immunity, but also in other sectors of immunology (e.g, autoimmunity and transplantation).
CD4⁻8⁻DCs, Tolerogenic, Regulatory T cells, Conversion, CpG, CD8⁺CD25⁺, pMHC I
Doctor of Philosophy (Ph.D.)