Search

Background: Tumor cell-derived exosomes (TEXs) have been widely used to induce antitumor immune responses in animal models and clinical trials. Similarly, leukemia cell-derived exosomes (LEXs) can induce antileukemia immune responses in animal models. However, the antileukemia immunity induced by LEXs is less effective, which may be due to an inadequate costimulatory capacity.Methods: In this study, we transduced L1210 leukemia cells with a lentiviral vector encoding two B7 costimulatory molecules (CD80, CD86) and obtained LEXs that highly expressed CD80 and CD86. The antileukemia immune response derived from these LEXs was examined in vitro and in vivo in animal models.Results: We found that B7 gene-modified LEXs, including LEX-CD80, LEX-CD86, and LEX-8086, could significantly boost the expression of CD80 and CD86 in dendritic cells (DCs) and promote the secretion of functional cytokines such as TNF-α and IL-12. Moreover, these B7 gene-modified LEXs, particularly LEX-CD8086, could effectively induce CD4+ T cell proliferation, Th1 cytokine secretion, and an antigen-specific anti-leukemia cytotoxic T lymphocyte (CTL) response. Additional animal studies indicated that immunization with B7 gene-modified LEXs, in particular LEX-CD8086, could significantly retard tumor growth compared to the control LEXnull group.Conclusions: This study sheds light on the feasibility of obtaining LEXs that overexpress costimulatory molecules via genetically modified leukemia cells, thereby enhancing their anti-leukemia immunity and providing a potential therapeutic strategy that contributes to leukemia immunotherapy.

Leukemia and lymphoma are common hematological malignancies in children and young adults, which pose a tremendous threat to the survival of these young patients worldwide, despite availability of various effective treatments. The Hippo pathway is a novel‑signaling pathway that regulates organ size, cell proliferation, apoptosis and tumorigenesis. The chief component of this pathway is the transducer yes‑associated protein (YAP) which is over‑expressed in numerous categories of tumors. However, little is known about the effect of YAP in hematological malignancies. In the present study, YAP expression was screened in several leukemia and lymphoma cell lines, and high YAP expression was demonstrated in Jurkat cells. To further unravel its effect on the biological behavior of Jurkat cells, lentivirus transduced short hairpin RNA (shRNA) technique was used to silence YAP. As expected, the YAP‑specific shRNA dramatically inhibited YAP expression at the mRNA and protein levels. Reduced leukemia cell proliferation and increased cell apoptosis were demonstrated in YAP knockdown Jurkat cells. It was also demonstrated that YAP knockdown resulted in deregulated expression of a cluster of downstream genes crucial to cell proliferation or apoptosis, including protein kinase B, B‑cell lymphoma 2 (BCL2) and BCL2 like protein 1. Consequently, the results of the present study established that suppression of YAP expression serves an important role in Jurkat cell proliferation and apoptosis, which may serve as a potential therapeutic target.

Background/Aims: Minimal residual leukemia cells (MRLs) are difficult to eradicate through traditional treatment and therefore remain to be a major threat to the long-term survival of leukemia patients. Tumor-derived exosomes (TEXs), which carry tumor associated antigens (TAA), may be a potential cell-free tumor vaccine for the specific eradication of MRLs. However, TEXs are intended to be less immunogenic due to exosomal TGF-β1. To further optimize the efficacy of TEX-based vaccines, we investigated whether exosomes from TGF-β1 silenced leukemia cells (LEXTGF-β1si) had an increased potential to induce a specific antitumor effect compared with non-modified exosomes. Methods: Exosomal TGF-β1 was downregulated via lentiviral shRNA silencing of TGF-β1 in leukemia cells. The characteristics of LEXTGF-β1si were determined via electron microscopy, western blot analysis, and flow cytometry. The antitumor effect of LEXTGF-β1si was evaluated by detecting the properties of LEXTGF-β1si-pulsed dendritic cells (DCs), CD4+ T-cell proliferation, Th1 cytokine secretion, specific CTL activity, and NK cell function. Moreover, to verify the superiority of LEXTGF-β1si immunization, LEXTGF-β1si was subcutaneously injected into DBA/2 mice: either followed by tumor challenge or tumor bearing. Results: The lentiviral shRNA silencing of TGF-β1 in parental leukemia cells successfully downregulated the TGF-β1 level in leukemia cell derived exosomes (LEX). LEXTGF-β1si was uptaken by DCs and was more potent in promoting DC function by upregulating the surface expression of costimulatory factors and MHC class II molecules, while inducing the secretion of IL-12p70 and TNF-α. Furthermore, immunization with LEXTGF-β1si facilitated CD4+ T-cell proliferation and Th1 cytokine secretion, and stimulated stronger specific cytotoxic lymphocyte (CTL) response and nature killer (NK) cell cytotoxicity more efficiently compared to non-modified LEX. In mice models, immunization with LEXTGF-β1si resulted in a more potent capability to inhibit tumor growth and to prolong survival, suggesting that LEXTGF-β1si was more effective in both protective and therapeutic antitumor tests than non-modified LEX. Conclusions: These data suggested that down-regulation of exosomal TGF-β1 effectively induced potent anti-tumor immunity. Our strategy of optimizing exosome vaccine may have promising potential for leukemia immunotherapy.

Aberrant over-expression of BCL-2 family proteins (BCL-2, BCL-xL, MCL-1) are associated with hematological malignancies. Antagonists of BCL-2 family proteins include BCL-2-selective inhibitor ABT-199, MCL-1-selective inhibitor A-1210477, BCL-xL-selective inhibitor A-1155463. In this study, we evaluated their potential inhibitory effectiveness. Our data showed that OCI-AML3 cells and U937 cells were resistant to BCL-2-selective inhibitor ABT-199 in vitro and in vivo, however, while OCI-AML3 cells were sensitive to MCL-1-selective inhibitor A-1210477 in vitro and in vivo, indicating that A-1210477 could counteract the resistance of AML cells to ABT-199 as a single agent in MCL-1-dependent AML cells. U-937 cell line and mouse model were resistant to A-1210477 or ABT-199, and expressed high level of BCL-xL, indicating that BCL-xL might play an important role in the resistance of A-1210477 or ABT-199. Besides, this study also showed that ABT-199 could synergize with A-1210477 in vitro or in vivo.

Although 1,25‑dihydroxyvitamin D3 (VD3) is regarded as a promising inducing agent for leukemia cell differentiation, it is not as effective an agent as all‑trans‑retinoic acid, and its usefulness is also limited by the adverse effects of hypercalcemia. The aim of the present study was to determine whether combining VD3 with adenanthin, a peroxiresoxin I (Prx I)‑targeting natural compound, improves the efficacy of VD3. Cell viability was assessed using a trypan blue exclusion assay and flow cytometry was used to evaluate the expression of cell surface markers, CD11b/CD14, and the level of reactive oxygen species (ROS). Wright's staining was used to examine morphological changes and RNA‑interference was used to knockdown Prx I and p65 gene expression. Protein expression was determined by western blot analysis. The results demonstrated that adenanthin markedly enhanced VD3‑induced cell differentiation of leukemia NB4 cells, as evidenced by the increased percentage of CD11b‑ and CD14‑positive cells, the mature morphology of the monocytes and the increased phagocytic ability. Consistent with these results, knockdown of Prx I, but not nuclear factor‑κB (p65), enhanced VD3‑induced cell differentiation. The combinatorial effects of adenanthin and VD3 were shown to be associated with the ROS‑CCAAT‑enhancer‑binding protein (C/EBP)β axis, since N‑acetylcysteine, a ROS scavenger, was able to abrogate the differentiation‑enhancing effects of adenanthin, and the knockdown of C/EBPβ also inhibited the combinatorial effects of adenanthin and VD3. In addition, co‑treatment with adenanthin and VD3 was able to induce differentiation in other non‑acute promyelocytic leukemia cells and primary leukemia cells. In conclusion, the results of the present study revealed a novel role for Prx I in VD3‑induced cell differentiation, and suggested that targeting Prx I may represent a novel strategy to enhance VD3‑induced leukemia cell differentiation.

Hemophilia A is an inherited X-linked recessive bleeding disorder caused by coagulant factor VIII (FVIII) deficiency. The conventional treatment involves the administration of recombinant human FVIII (rhFVIII) preparations. In this study, the mammary gland ‘bioreactor’ is designed to specifically and efficiently express a foreign protein hFVIII in the mammary glands of transgenic mice. We constructed a P1A3-hFVIIIBD vector directed by the mammary gland-specific P1A3 promoter, and transiently transfected HC11 cells and mouse mammary glands with P1A3-hFVIIIBD or CMV-hFVIIIBD vectors directed by a ubiquitous cytomegalovirus (CMV) promoter, respectively. We also generated P1A3-hFVIIIBD and CMV-hFVIIIBD transgenic mice by microinjection, respectively. Our data indicated that both vectors effectively expressed hFVIIIBD in HC11 cells at the transcription level, and hFVIIIBD protein was efficiently expressed in mouse milk after the injection of the hFVIIIBD vectors into mouse mammary glands during lactation. In both CMV-hFVIIIBD and P1A3-hFVIIIBD transgenic mice, hFVIIIBD proteins were efficiently expressed in the mammary glands at the mRNA and protein levels. No significant difference was observed in hFVIIIBD levels between the CMV-hFVIIIBD and P1A3-hFVIIIBD transgenic mice (P > 0.05). However, the activity of hFVIII in CMV-directed transgenic mice was slightly higher than that in P1A3-directed transgenic mice (P

Tumor-derived exosomes (TEX) can induce a specific antitumor immune response and have been developed as a promising tumor vaccine. Despite promising preclinical data, TEX exhibit relatively low efficacy and limited clinical benefit in clinical trials. In the present study, we investigated whether exosomes from the TGF-β1 silenced L1210 cells (LEXTGF-β1si) can enhance the efficacy of DC-based vaccines. We silenced TGF-β1 in L1210 cells with a lentiviral shRNA vector and prepared the LEXTGF-β1si. It was shown that LEXTGF-β1si can significantly decrease TGF-β1 expression of dendritic cells (DC) and effectively promote their maturation and immune function. In addition, DC pulsed with LEXTGF-β1si (DCLEX-TGF-β1si) more effectively promoted CD4+ T cell proliferation in vitro and Th1 cytokine secretion and induced tumor-specific CTL response. This response was higher in potency compared to that noted by the other two formulations. Moreover, DCLEX-TGF-β1si inhibited tumor growth more efficiently than other formulations did as the preventive or therapeutic tumor vaccine. Accordingly, these findings revealed that DCLEX-TGF-β1si induced a more potent antigen-specific anti-leukemic immunity than DC pulsed with exosomes from non-manipulated L1210 cells. This indicated that the targeting of DC by LEXTGF-β1si may be used as a promising approach for leukemia immunotherapy.

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.

Exosomes (EXOs) are nanometer-sized membrane vesicles secreted from epithelial and hematopoietic cells. They display a spectrum of molecules involved in immune responses and signal transductions. Previous studies showed that tumor antigen-loaded dendritic cell (DC)- and tumor cell-derived EXOs (Dexo and Texo) induce tumor antigen-specific CD8(+) cytotoxic T-lymphocyte responses and antitumor immunity in experimental animal models and human clinical trials. This review will present the main biologic features of Dexo and Texo as cell-free cancer vaccines with emphasis on their immunostimulatory properties and their potential efficacy in cancer immunotherapy.

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.

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).

The CXC chemokine SDF-1 has been characterized as a T-cell chemoattractant both in vitro and in vivo. To determine whether SDF-1 expression within tumors can influence tumor growth, we transfected an expression vector pCI-SDF-1 for SDF-1 into J558 myeloma cells and tested their ability to form tumors in BALB/c. Production of biologically active SDF-1 (1.2 ng/mL) was detected in the culture supernatants of cells transfected with the expression vector pCI-SDF-1. J558 cells gave rise to a 100% tumor incidence, whereas SDF-1-expressing J558/SDF-1 tumors invariably regressed in BALB/c mice and became infiltrated with CD4(+) and CD8(+) T cells. Regression of the J558/SDF-1 tumors was dependent on both CD4(+) and CD8(+) T-cells. Our data also indicate that TIT cells containing both CD4(+) and CD8(+) T-cells within J558/SDF-1 tumors express the SDF-1 receptor CXCR4, and that SDF-1 specifically chemoattracts these cells in vitro. Furthermore, immunization of mice with engineered J558/SDF-1 cells elicited the most potent protective immunity against 0.5 x 10(6) cells J558 tumor challenge in vivo, compared to immunization with the J558 alone, and this antitumor immunity mediated by J558/SDF-1 tumor cell vaccination in vivo appeared to be dependent on CD8(+) CTL. Thus, SDF-1 has natural adjuvant activities that may augment antitumor responses through their effects on T-cells and thereby could be important in gene transfer immunotherapies for some cancers.

The P815 and P198 cell lines are clonally related mouse mastocytoma cell lines. They differ in their biologic behavior in that P815 is a progressive tumor cell line, whereas P198 is a regressive one. These cell lines have been extensively used as models for the study of tumor-host relationships and tumor immunology. Although some of their biological properties have been well documented, the molecular mechanisms underlying tumor progression or regression have not been completely elucidated. In this study, we characterized the growth behavior and immunophenotype of these two cell lines, and analyzed their gene profiles using a complementary deoxynucleic acid (cDNA) microarray composed of 514 immunologically relevant genes. Our data showed that the two cell lines exhibited quite dissimilar and contrasting growth characteristics when inoculated into syngeneic mice. P815 tumors grew unremittingly, while P198 tumors gradually regressed. From a molecular viewpoint, P815 cells showed a higher expression of genes promoting tumor growth, such as IGF-1, IL-8R, FGFR1, VEGF-A, and VEGF-B. On the other hand, P198 tumor cells expressed CD11b and CD80, which favor the recruitment of lymphocytes and antigen-presenting cells (APCs), as well as the elicitation of antitumor immunity. P198 tumor cells also depicted a higher expression of genes inhibiting tumor growth, such as TNF-alpha, SOCS-1, CIS1, 4-1BB, and GDF-10. In conclusion, our results contribute further information in the understanding of the molecular mechanisms associated with the regression and progression of P815 and P198 tumor cells.

Dendritic cells (DCs) and tumor cell-derived exosomes have been used to develop antitumor vaccines. However, the biological properties and antileukemic effects of leukemia cell-derived exosomes (LEXs) are not well described. In this study, the biological properties and induction of antileukemic immunity of LEXs were investigated using transmission electron microscopy, western blot analysis, cytotoxicity assays, and animal studies. Similar to other tumor cells, leukemia cells release exosomes. Exosomes derived from K562 leukemia cells (LEXK562) are membrane-bound vesicles with diameters of approximately 50–100 μm and harbor adhesion molecules (e.g., intercellular adhesion molecule-1) and immunologically associated molecules (e.g., heat shock protein 70). In cytotoxicity assays and animal studies, LEXs-pulsed DCs induced an antileukemic cytotoxic T-lymphocyte immune response and antileukemic immunity more effectively than did LEXs and non-pulsed DCs (P

Tumor cell-derived exosomes (TEX) have been widely used to induce antitumor immune responses in animal models and clinical trials. However, the efficiency of the antitumor immunity that is induced by TEX is still relatively weak. In this study, we compared the antitumor immunities between EG7 tumor cell-derived exosomes (EXO(EG7)) and EXO(EG7)-targeted dendritic cells (DC(EXO)). We found that EXO(EG7) harbored OVA and peptide major histocompatibility complex I (pMHC-I), which were expressed on its parental EG7 tmor cells, and they could transfer OVA and pMHC-I to dendritic cells (DCs) in vitro. DC(EXO) could more efficiently induce antitumor immunity than EXO(EG7). In addition, we showed that the immune stimulatory effects of EXO(EG7) were dependent on the host DCs and, whereas those of DC(EXO) were not, indicating the important role of the host DCs in TEX vaccines. Taken together, TEX-targeted DCs may be more effective for EXO-based vaccines for the induction of antitumor immunity.

One of the major obstacles in human epidermal growth factor receptor 2 (HER2)-specific trastuzumab antibody immunotherapy of HER2-positive breast cancer is the development of trastuzumab resistance, warranting the search for other therapeutic strategies. Using mouse models, we previously demonstrated that ovalbumin (OVA)-specific dendritic cell (DC)-released exosome (EXOOVA)-targeted CD4(+) T cell-based (OVA-TEXO) vaccine stimulates efficient cytotoxic T lymphocyte (CTL) responses via exosomal peptide/major histocompatibility complex (pMHC)-I, exosomal CD80 and endogenous IL-2 signaling; and long-term CTL memory by means of via endogenous CD40L signaling. In this study, using two-photon microscopy, we provide the first visual evidence on targeting OVA-TEXO to cognate CD8(+) T cells in vivo via exosomal pMHC-I complex. We prepared HER2/neu-specific Neu-TEXO and HER2-TEXO vaccines using adenoviral vector (AdVneu and AdVHER2)-transfected DC (DCneu and DCHER2)-released EXOs (EXOneu and EXOHER2), and assessed their stimulatory effects on HER2/neu-specific CTL responses and antitumor immunity. We demonstrate that Neu-TEXO vaccine is capable of stimulating efficient neu-specific CTL responses, leading to protective immunity against neu-expressing Tg1-1 breast cancer in all 6/6 transgenic (Tg) FVBneuN mice with neu-specific self-immune tolerance. We also demonstrate that HER2-TEXO vaccine is capable of inducing HER2-specific CTL responses and protective immunity against transgene HLA-A2(+)HER2(+) BL6-10A2/HER2 B16 melanoma in 2/8 double Tg HLA-A2/HER2 mice with HER2-specific self-immune tolerance. The remaining 6/8 mice had significantly prolonged survival. Furthermore, we demonstrate that HER2-TEXO vaccine stimulates responses of CD8(+) T cells capable of not only inducing killing activity to HLA-A2(+)HER2(+) BL6-10A2/HER2 melanoma and trastuzumab-resistant BT474A2 breast cancer cells in vitro but also eradicating 6-day palpable HER2(+) BT474A2 breast cancer (3-4 mm in diameter) in athymic nude mice. Therefore, the novel T cell-based HER2-TEXO vaccine may provide a new therapeutic alternative for women with HER2(+) breast cancer, especially for trastuzumab-resistant HER2(+) breast cancer patients.

CD4+ helper T (Th) cells play crucial role in priming, expansion and survival of CD8+ cytotoxic T lymphocytes (CTLs). However, how CD4+ Th cell's 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 (DC(OVA)) stimulation, and then stimulate OVA-specific CD8+ CTL responses in C57BL/6 mice. In this study, we further investigated CD4+ Th cell's 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 DC(OVA)-activated CD4+ Th cells (3 x 10(6) 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 x 10(6) 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.

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.

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 naive 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 cell's 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.

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 (DC(OVA))-derived EXO (EXO(OVA)) 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 DC(OVA). EXO(OVA) 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 EXO(OVA), which were referred to as mDC(EXO), expressed a higher level of pMHC I, MHC II, and costimulatory CD40, CD54 and CD80 than DC(OVA). The mDC(EXO) 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 EXO(OVA) and DC(OVA). In addition, mDC(EXO) 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.

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.

Because dendritic cell (DC)-derived exosomes (EXO) harbor many important DC molecules involved in inducing immune responses, EXO-based vaccines have been extensively used to induce antitumor immunity in different animal tumor models. However, it is not clear which route of EXO administration can induce more efficient antitumor immune responses. In this study, we compared the antitumor immunity derived from EXO vaccine by way of the two common administration routes, the subcutaneous (s.c.) and the intradermal (i.d.) administrations. Our data showed that the i.d. EXO administration resulted in more EXO-absorbed DC migrating into the T-cell areas of draining lymph nodes than the s.c. administration. Interestingly, the i.d. EXO administration also resulted in an enhanced ovalbumin (OVA)-specific CD8(+) T-cell proliferation and CD8(+) CTL effector responses in vivo, compared to the s.c. administration. Similarly, compared to the s.c. vaccination, the i.d. vaccination induced stronger antitumor immunity in the animal tumor model. Therefore, the i.d. EXO vaccination is superior to the s.c. one and should be considered when EXO-based vaccine is designed.

Implantation of microencapsulated engineered cells secreting molecules with antineoplastic properties into tumors is a novel approach to cancer gene therapy. In this study, we constructed an engineered tumor cell line, VkCk/RM4-TNF-alpha, which secreted RM4/TNF-alpha fusion protein containing the chimeric antitumor antibody, F(ab')2 (RM4), recognizing the tumor antigen TAG72, as well as the TNF-alpha moiety. The engineered cells were encapsulated into microencapsules. The RM4/TNF-alpha fusion protein secreted by encapsulated VkCk/RM4-TNF-alpha cells could be diffused through the microencapsule membrane into the supernatant and exert a cytotoxic effect on L929 cells in vitro. The antigen-specific binding-reactivity of RM4/TNF-alpha for the TAG72 antigen was confirmed by immunohistochemical staining of rat LMCR tumor cells which expressed TAG72 antigen. Implantation of microencapsules containing VkCk/RM4-TNF-alpha cells into LMCR tumors in rats induced tumor regression as a result of tumor necrosis formation. Taken together, these data suggest that microencapsulation of recombinant tumor cells secreting antibody/cytokine fusion protein might be an alternative approach in the treatment of cancers.

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.

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.

Background Exosomes secreted by tumor cells contain specific antigens that may have immunotherapeutic purposes. The aim of this study was to characterize the proteomic content of lymphoma cell-derived exosomes (LCEXs). Methods In this study, exosomes derived from Raji cells (EXORaji) were purified and proteins of EXORaji were separated by one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis. Protein bands were identified by mass spectrometry. The protein components of EXORaji were analyzed using shotgun technology, and the function proteins of EXORaji were defined and described using the Gene Ontology (GO) database and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Results A total of 197 proteins were identified in EXORaji; 139 proteins were also identified in Raji cells, showing an overlap of 70.56% of the total proteins in EXORaji. Interestingly, the remaining 58 proteins were unique to EXORaji. The GO database and KEGG were used to define and describe the function of proteins. The data showed that some important proteins involved in antigen procession and presentation as well as cell migration and adhesion were also identified in EXORaji, such as MHC-I and II, HSC70, HSP90, and ICMA-1. Conclusions LCEXs express a discrete set of proteins involved in antigen presentation and cell migration and adhesion, suggesting that LCEXs play an important role in the regulation of immunity and interaction between lymphoma cells and their microenvironment. LCEXs harbor most of the proteins of lymphoma cells and could be one of the sources of lymphoma-associated antigens for immunotherapeutic purposes.