Siguo Hao

Mature dendritic cells pulsed with exosomes stimulate efficient cytotoxic T‐lymphocyte responses and antitumour immunity

Exosomes (EXO) derived from dendritic cells (DC), which express major histocompatibility complex (MHC) and costimulatory molecules, have been used for antitumour vaccines. However, they are still less effective by showing only prophylatic immunity in animal models or very limited immune responses in clinical trials. In this study, we showed that ovalbumin (OVA) protein‐pulsed DC (DCOVA)‐derived EXO (EXOOVA) displayed MHC class I–OVA I peptide (pMHC I) complexes, CD11c, CD40, CD80, CCR7, DEC205, Toll‐like receptor 4 (TLR4), TLR9, MyD88 and DC‐SIGN molecules, but at a lower level than DCOVA. EXOOVA can be taken up by DC through LFA‐1/CD54 and C‐type lectin/mannose (glucosamine)‐rich C‐type lectin receptor (CLR) interactions. Mature DC pulsed with EXOOVA, which were referred to as mDCEXO, expressed a higher level of pMHC I, MHC II, and costimulatory CD40, CD54 and CD80 than DCOVA. The mDCEXO could more strongly stimulate OVA‐specific CD8+ T‐cell proliferation in vitro and in vivo, and more efficiently induce OVA‐specific cytotoxic T‐lymphocyte responses, antitumour immunity and CD8+ T‐cell memory in vivo than EXOOVA and DCOVA. In addition, mDCEXO could also more efficiently eradicate established tumours. Therefore, mature DC pulsed with EXO may represent a new, highly effective DC‐based vaccine for the induction of antitumour immunity.

CD8+ Cytotoxic T-APC Stimulate Central Memory CD8+ T Cell Responses via Acquired Peptide-MHC Class I Complexes and CD80 Costimulation, and IL-2 Secretion

We previously showed that naive CD4+ Th cells acquire peptide-MHC class I (pMHC I) and costimulatory molecules from OVA-pulsed dendritic cells (DCOVA), and act as Th-APCs in stimulation of CD8+CTL responses. In this study, we further demonstrated that naive CD8+ cytotoxic T (Tc) cells also acquire pMHC I and costimulatory CD54 and CD80 molecules by DCOVA stimulation, and act as Tc-APC. These Tc-APC can play both negative and positive modulations in antitumor immune responses by eliminating DCOVA and neighboring Tc-APC, and stimulating OVA-specific CD8+ central memory T responses and antitumor immunity. Interestingly, the stimulatory effect of Tc-APC is mediated via its IL-2 secretion and acquired CD80 costimulation, and is specifically targeted to OVA-specific CD8+ T cells in vivo via its acquired pMHC I complexes. These principles could be applied to not only antitumor immunity, but also other immune disorders (e.g., autoimmunity).

CD4+ Th1 cells promote CD8+ Tc1 cell survival, memory response, tumor localization and therapy by targeted delivery of interleukin 2 via acquired pMHC I complexes

The cooperative role of CD4+ helper T (Th) cells has been reported for CD8+ cytotoxic T (Tc) cells in tumor eradication. However, its molecular mechanisms have not been well elucidated. We have recently demonstrated that CD4+ Th cells can acquire major histocompatibility complex/peptide I (pMHC I) complexes and costimulatory molecules by dendritic cell (DC) activation, and further stimulate naïve CD8+ T cell proliferation and activation. In this study, we used CD4+ Th1 and CD8+ Tc1 cells derived from ovalbumin (OVA)‐specific T cell receptor (TCR) transgenic OT II and OT I mice to study CD4+ Th1 cells help effects on active CD8+ Tc1 cells and the molecular mechanisms involved in CD8+ Tc1‐cell immunotherapy of OVA‐expressing EG7 tumors. Our data showed that CD4+ Th1 cells with acquired pMHC I by OVA‐pulsed DC (DCOVA) stimulation are capable of prolonging survival and reducing apoptosis formation of active CD8+ Tc1 cells in vitro, and promoting CD8+ Tc1 cell tumor localization and memory responses in vivo by 3‐folds. A combined adoptive T‐cell therapy of CD8+ Tc1 with CD4+ Th1 cells resulted in regression of well‐established EG7 tumors (5 mm in diameter) in all 10/10 mice. The CD4+ Th1’s help effect is mediated via the helper cytokine IL‐2 specifically targeted to CD8+ Tc1 cells in vivo by acquired pMHC I complexes. Taken together, these results will have important implications for designing adoptive T‐cell immunotherapy protocols in treatment of solid tumors.

Nonspecific CD4+ T cells with uptake of antigen-specific dendritic cell-released exosomes stimulate antigen-specific CD8+ CTL responses and long-term T cell memory

Dendritic cell (DC) and DC‐derived exosomes (EXO) have been used extensively for tumor vaccination. However, its therapeutic efficiency is limited to only production of prophylactic immunity against tumors. T cells can uptake DC‐released EXO. However, the functional effect of transferred exosomal molecules on T cells is unclear. In this study, we demonstrated that OVA protein‐pulsed DC‐derived EXO (EXOOVA) can be taken up by Con A‐stimulated, nonspecific CD4+ T cells derived from wild‐type C57BL/6 mice. The active EXO‐uptaken CD4+ T cells (aTEXO), expressing acquired exosomal MHC I/OVA I peptide (pMHC I) complexes and costimulatory CD40 and CD80 molecules, can act as APCs capable of stimulating OVA‐specific CD8+ T cell proliferation in vitro and in vivo and inducing efficient CD4+ Th cell‐independent CD8+ CTL responses in vivo. The EXOOVA‐uptaken CD4+ aTEXO cell vaccine induces much more efficient CD8+ T cell responses and immunity against challenge of OVA‐transfected BL6‐10 melanoma cells expressing OVA in wild‐type C57BL/6 mice than EXOOVA. The in vivo stimulatory effect of the CD4+ aTEXO cell to CD8+ T cell responses is mediated and targeted by its CD40 ligand signaling/acquired exosomal CD80 and pMHC I complexes, respectively. In addition, CD4+ aTEXO vaccine stimulates a long‐term, OVA‐specific CD8+ T cell memory. Therefore, the EXOOVA‐uptaken CD4+ T cells may represent a new, effective, EXO‐based vaccine strategy in induction of immune responses against tumors and other infectious diseases.

Novel Exosome-Targeted CD4+ T Cell Vaccine Counteracting CD4+ 25+ Regulatory T Cell-Mediated Immune Suppression and Stimulating Efficient Central Memory CD8+ CTL Responses

T cell-to-T cell Ag presentation is increasingly attracting attention. In this study, we demonstrated that active CD4+ T (aT) cells with uptake of OVA-pulsed dendritic cell-derived exosome (EXOOVA) express exosomal peptide/MHC class I and costimulatory molecules. These EXOOVA-uptaken (targeted) CD4+ aT cells can stimulate CD8+ T cell proliferation and differentiation into central memory CD8+ CTLs and induce more efficient in vivo antitumor immunity and long-term CD8+ T cell memory responses than OVA-pulsed dendritic cells. They can also counteract CD4+25+ regulatory T cell-mediated suppression of in vitro CD8+ T cell proliferation and in vivo CD8+ CTL responses and antitumor immunity. We further elucidate that the EXOOVA-uptaken (targeted)CD4+ aT cell’s stimulatory effect is mediated via its IL-2 secretion and acquired exosomal CD80 costimulation and is specifically delivered to CD8+ T cells in vivo via acquired exosomal peptide/MHC class I complexes. Therefore, EXO-targeted active CD4+ T cell vaccine may represent a novel and highly effective vaccine strategy for inducing immune responses against not only tumors, but also other infectious diseases.

CD4 T cells stimulate memory CD8 T cell expansion via acquired pMHC I complexes and costimulatory molecules, and IL-2 secretion

The rapid and efficient expansion of CD8+ memory T cells after the second encounter with a pathogen constitutes a hallmark trait of adaptive immunity. Yet, the contribution of CD4+ T cells to the expansion of memory CD8+ T cells remains the subject of controversy. Here, we show that, antigen‐specific CD4+ T cells, once activated by dendritic cells (DC) in vitro, have the capacity to stimulate expansion of memory CD8+ T cells in vivo. The memory CD8+ T cell expansion triggered by active CD4+ T cells are mediated through DC‐derived MHC I/peptide complexes and CD80 molecules displayed on the active CD4+ T cells, with the involvement of IL‐2 secreted by the active CD4+ T cells. These results highlight a previously undescribed role of active CD4+ T cells in triggering expansion of memory CD8+ T cells.

IL-10 Has A Distinct Immunoregulatory Effect on Naive and Active T Cell Subsets

Interleukin-10 (IL-10) has been identified as a key immunomodulatory cytokine on T cells. However, both immunosuppressive and immunostimulatory effects of IL-10 on T cells also have been reported. The discrepancy between these in vitro effects of IL-10 may be due to the different T cells (naive vs. active or resting active T cells) used under various experimental conditions in these studies. Therefore, it is necessary to clearly define the IL-10 effect on T cell subsets in their different statuses. In this study, we used a molecularly defined T cell system, the ovalbumin (OVA)-specific CD4(+) and CD8(+) T cells from transgenic OT-I and OT-II mice expressing OVA-specific T cell receptor (TCR). We investigated the effect of IL-10 on these OVA-specific T cell subsets in their different statuses (i.e., naive and active T cells). Our data demonstrate that IL-10 has distinct immunoregulatory effects on naive and active T cell subsets. IL-10 inhibits active CD4(+) T cell proliferation, whereas it stimulates and suppresses active CD8(+) T cell proliferation and cytotoxicity, respectively. IL-10-treated dendritic cells (DCs) stimulate anergic cytotoxic T lymphocyte-associated molecule-4 (CTLA)-4-expressing CD4(+) T cell responses possibly through downregulation of major histocompetibility complex (MHC) class II and costimulatory molecule expression on DCs. The anergic CD4(+) T cells suppress T cell proliferation mainly through a CTLA-4-mediated pathway. The distinct role of IL-10 on T cell subsets may be useful in designing T cell-based immunotherapy of cancer and infectious diseases.

Antigen Specificity Acquisition of Adoptive CD4+ Regulatory T Cells via Acquired Peptide-MHC Class I Complexes

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.

Active CD4+ helper T cells directly stimulate CD8+ cytotoxic T lymphocyte responses in wild-type and MHC II gene knockout C57BL/6 mice and transgenic RIP-mOVA mice expressing islet b-cell ovalbumin antigen leading to diabetes

CD4+ helper T (Th) cells play crucial role in priming, expansion and survival of CD8+ cytotoxic T lymphocytes (CTLs). However, how CD4+ Th cells help is delivered to CD8+ T cells in vivo is still unclear. We previously demonstrated that CD4+ Th cells can acquire ovalbumin (OVA) peptide/major histocompatibility complex (pMHC I) and costimulatory CD80 by OVA-pulsed DC (DCOVA) stimulation, and then stimulate OVA-specific CD8+ CTL responses in C57BL/6 mice. In this study, we further investigated CD4+ Th cells effect on stimulation of CD8 CTL responses in major histocompatibility complex (MHC II) gene knockout (KO) mice and transgenic rat insulin promoter (RIP)-mOVA mice with moderate expression of self OVA by using CD4+ Th cells or Th cells with various gene deficiency. We demonstrated that the in vitro DCOVA-activated CD4+ Th cells (3 × 106 cells/mouse) can directly stimulate OVA-specific CD8+ T-cell responses in wild-type C57BL/6 mice and MHC II gene KO mice lacking CD4+ T cells. A large amount of CD4+ Th cells (12 × 106 cells/mouse) can even overcome OVA-specific immune tolerance in transgenic RIP-mOVA mice, leading to CD8+ CTL-mediated mouse pancreatic islet destruction and diabetes. The stimulatory effect of CD4+ Th cells is mediated by its IL-2 secretion and CD40L and CD80 costimulations, and is specifically delivered to OVA-specific CD8+ T cells in vivo via its acquired pMHC I complexes. Therefore, the above elucidated principles for CD4+ Th cells will have substantial implications in autoimmunity and antitumor immunity, and regulatory T-cell-dependent immune suppression.

Genetically Engineered Myeloma Cell Vaccine

Tumor cells engineered to express immunogenes have been used for cancer vaccines to induce antitumor immunity and to study the antitumor immune mechanisms derived from immunogene expression. In this chapter, we describe the design and methods for cloning a cDNA gene coding for the mouse CD40L molecule and for construction of the expression vector pcDNA-CD40L, as well as the methods for generation of engineered myeloma cells J558/CD40L expressing CD40 ligand. We also demonstrate that the engineered J558/CD40L tumor cells lose their tumorigenicity in syngeneic mice, and that the inoculation of J558/CD40L tumor cells further leads to protective immunity against wild-type J558 tumors.