Yongqing Liu

A New Dynamic Model of CD8+ T Effector Cell Responses via CD4+ T Helper-Antigen-Presenting Cells

A long-standing paradox in cellular immunology has been the conditional requirement for CD4+ Th cells in priming of CD8+ CTL responses. We propose a new dynamic model of CD4+ Th cells in priming of Th-dependent CD8+ CTL responses. We demonstrate that OT II CD4+ T cells activated by OVA-pulsed dendritic cells (DCOVA) are Th1 phenotype. They acquire the immune synapse-composed MHC II/OVAII peptide complexes and costimulatory molecules (CD54 and CD80) as well as the bystander MHC class I/OVAI peptide complexes from the DCOVA by DCOVA stimulation and thus also the potential to act themselves as APCs. These CD4+ Th-APCs stimulate naive OT I CD8+ T cell proliferation through signal 1 (MHC I/OVAI/TCR) and signal 2 (e.g., CD54/LFA-1 and CD80/CD28) interactions and IL-2 help. In vivo, they stimulate CD8+ T cell proliferation and differentiation into CTLs and induce effective OVA-specific antitumor immunity. Taken together, this study demonstrates that CD4+ Th cells carrying acquired DC Ag-presenting machinery can, by themselves, efficiently stimulate CTL responses. These results have substantial implications for research in antitumor and other aspects of immunity.

Engineered fusion hybrid vaccine of IL-4 gene-modified myeloma and relative mature dendritic cells enhances antitumor immunity

Dendritic cell (DC)-tumor fusion hybrid vaccine which facilitates antigen presentation represents a new powerful strategy in cancer therapy. In the present study, we investigated the antitumor immunity derived from vaccination of fusion hybrids between wild-type J558 or engineered J558-IL-4 myeloma cells secreting cytokine interleukin-4 (IL-4) and immature DCs (DC(IMAT)) or relative mature DCs (DC(RMAT)). DC(RMAT) displayed an up-regulated expression of immune molecules (Ia(d), CD40, CD54, CD80 and CD86) and certain cytokines/chemokines, and enhanced ability of allogeneic T cell stimulation when compared to DC(IMAT). These DCs were fused with myeloma cells by polyethylene glycol (PEG). The fusion efficiency was approximately 20%. Our data showed that immunization of C57BL/6 mice with DC(RMAT)/J558 hybrids induced protective immunity against a high dose of J558 tumor challenge (1x10(6) cells) in 3 out of 10 immunized mice, compared with no protection seen in mice immunized with DC(IMAT)/J558 hybrids. Furthermore, immunization of mice with engineered DC(RMAT)/J558-IL-4 hybrids elicited stronger J558 tumor-specific cytotoxic T lymphocyte (CTL) responses in vitro and induced more efficient protective immunity (10/10 mice; tumor free) against J558 tumor challenge in vivo than DC(RMAT)/J558 hybrid vaccines. The results demonstrate the importance of DC maturation in DC-tumor hybrid vaccines and indicate that the engineered fusion hybrid vaccines which combine gene-modified tumor and DC vaccines may be an attractive strategy for cancer immunotherapy.

Adenovirus-mediated CD40 ligand gene-engineered dendritic cells elicit enhanced CD8 + cytotoxic T-cell activation and antitumor immunity

CD40L, the ligand for CD40 on dendritic cells (DCs), plays an important role in their activation and is essential for induction of antigen-specific T-cell responses. In the present study, we investigated the efficacy of antitumor immunity induced by vaccination with DCs engineered to express CD40L and pulsed with Mut1 tumor peptide. Our data show that transfection of DCs with recombinant adenovirus AdV-CD40L resulted in activation of DCs with up-regulated expression of proinflammatory cytokines (IL-1β and IL-12), chemokines (RANTES, IP-10, and MIP-1α), and immunologically important cell surface molecules (CD54, CD80, and CD86). Our data also demonstrate that DCs transfected with AdV-CD40L (DCCD40L) are able to stimulate enhanced allogeneic T-cell proliferation and Mut1-specific CD8+ cytotoxic T-cell responses in vitro. Vaccination of mice with Mut1 peptide-pulsed control virus–transfected DC (DCpLpA) could only protect mice from challenge of a low dose (0.5×105 cells per mouse, 8/8 mice), but not a high dose (3×105 cells per mouse, 0/8 mice) of 3LL tumor cells. However, vaccination of Mut1 peptide-pulsed AdV-CD40L–transfected DCCD40L induced an augmented antitumor immunity in vivo by complete protection of mice (8/8) from challenge of both low and high doses of 3LL tumor cells. Thus, DCs engineered to express CD40L by adenovirus-mediated CD40 ligand gene transfer may offer a new strategy in production of DC cancer vaccines.

Synergistic effect of adoptive T-cell therapy and intratumoral interferon γ-inducible protein-10 transgene expression in treatment of established tumors

The lack of efficient T-cell infiltration of tumors is a major obstacle to successful adoptive T-cell therapy. We have previously shown that transplanted SP2/0 myeloma tumors engineered to express lymphotactin invariably induced tumor regress mediated by SP2/0 tumor-specific T cells. Herein, we further systemically characterize these activated T cells and investigate their therapeutic efficacy, either alone or with the chemokine interferon gamma (IFN-gamma)-inducible protein-10 (IP-10) gene therapy. Following stimulation with SP2/0 cells, these activated T cells were CD25(+)FasL(+) L-selectin(low), expressed CXCR3 receptor and were chemoattracted by IP-10 in vitro. They comprised 64% CD4(+) Th1 and 36% CD8(+) Tc1 cells, both of which expressed IFN-gamma, perforin, and TNF-alpha, but not IL-4. The activated T cells were strongly cytotoxic for SP2/0 tumor cells (79% specific killing; E:T ratio, 50), mainly via perforin-mediated pathway. Cell tracking using labeled T cells confirmed that these T cells infiltrated better into the IP-10-expressing tumors than non-IP-10-expressing ones. In vivo, combined intratumoral IP-10 gene transfer and adoptive T-cell immunotherapy for well-established SP2/0 tumors eradicated the tumors in 7 of the 8 mice. Control or IP-10 adenoviral treatments by themselves neither alter the lethal outcome for tumor-bearing mice nor did T-cell therapy by itself, although the latter two treatments did slow its time-frame. Taken together, our data provide solid evidence of a potent synergy between adoptive T-cell therapy and IP-10 gene transfer into tumor tissues, which culminated in the eradication of well-established tumor masses.

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.

Intratumoral coinjection of two adenoviral vectors expressing functional interleukin-18 and inducible protein-10, respectively, synergizes to facilitate regression of established tumors.

We have constructed two recombinant adenoviral vectors AdVIP-10 and AdVIL-18 expressing the functional chemokine IFN-γ inducible protein (IP)-10 and cytokine interleukin (IL)-18, respectively. Injection of either AdVIP-10 or AdVIL-18 subcutaneously into tumor nodules derived from the J558 murine myeloma cell line delayed some tumor growth but it was not curative in all cases. Coinjection of these two vectors at the same tumor nodule not only significantly suppressed the tumor growth, but also cured established tumors in 8 of 10 (80% tumor free) mice. The latter treatment stimulated T-cell infiltration into tumors in association with tumor necrosis formation, induced a type 1 immune response and induced the activation of J558 tumor–specific cytotoxic T lymphocytes. Moreover, the antitumor activity of IP-10 and IL-18 combined gene therapy was significantly diminished in mice with depletion of either CD4+ (50% tumor free) or CD8+ (40% tumor free) T cells, and completely lost (0% tumor free) in T cell–deficient nude and IFN-γ knockout mice, indicating the critical roles of T cells and IFN-γ in this therapeutical model. Taken together, the findings of this study demonstrate that the combined use of two adenoviral vectors expressing IP-10 and IL-18, respectively, synergize to facilitate regression of established tumors. These observations also suggest the potential use of double-recombinant adenoviral vectors expressing chemokines and immunomodulatory cytokines in cancer gene therapy.

Tumor-Infiltrating Dendritic Cell Subsets of Progressive or Regressive Tumors Induce Suppressive or Protective Immune Responses

Tumor-infiltrating dendritic cells (TID) have an ambivalent role in regulation of tumor regression or growth. However, their precise natures and molecular mechanisms have not been elucidated. In this study, we studied TIDs recruited in progressive P815 and regressive P198 tumors of the same origin. Our data showed that P815 tumors contained CD4+ 8+ and CD4- 8- TID815 subsets, whereas P198 tumors contained CD4+ 8+ and CD4+ 8- TID198 subsets. They similarly stimulate allogeneic T cell proliferation and have nitric oxide-mediated cytotoxicity to tumor cells with an exception of CD4- 8- TID815 with less efficiency. The newly identified fourth CD4+ 8+ TID815 or TID198 subset and the CD4+ 8- TID198 all express high levels of IFN-gamma and interleukin (IL)-6, whereas CD4- 8- TID815 secrete a marked level of transforming growth factor-beta. Vaccination of mice with P815 tumor lysate-pulsed CD4+ 8+ TID815 or TID198 and CD4+ 8- TID198 induced IFN-gamma-secreting Th1 and effective CTL responses leading to protective immunity against P815 tumor, whereas CD4- 8- TID815 stimulated IL-10-expressing Tr1 responses leading to immune suppression. Transfer of CD4+ Tr1 cells obtained from CD4- 8- TID815-immunized wild-type, but not IL-10(-/-) mice, into CD4+ 8+ TID815 immunized mice abolished otherwise inevitable development of antitumor immunity. Taken together, our findings provide an important insight into immunologic alterations in progressive and regressive tumors and an implication for dendritic cell-based approaches in the design of cancer vaccines.

Dendritic cells engineered to express the Flt3 ligand stimulate type I immune response, and induce enhanced cytoxic T and natural killer cell cytotoxicities and antitumor immunity

Tumor antigen presentation by dendritic cells (DCs) to T cells in lymphoid organs is crucial for induction of antitumor immune responses. Fms‐like tyrosine kinase 3 ligand (Flt3L) is a regulator of hematopoietic cell development.

Intratumoral administration of immature dendritic cells following the adenovirus vector encoding CD40 ligand elicits significant regression of established myeloma.

Our previous study showed that J558 myeloma cells engineered CD40L lost their tumorigenicity in syngeneic mice, and the inoculation of J558/CD40L tumor cells further led to the protective immunity against wild tumors. In the present study, we investigated whether the vaccine can exert more efficient antitumor immunity by combination with adenovirus mediated CD40L gene therapy and immature dendritic cells (iDCs). The results demonstrated that intratumoral administration of iDCs 2 days after AdVCD40L injection, not only significantly suppressed the tumor growth, but also eradiated the established tumors in 40% of the mice. The potent antitumor effect produced by the combination therapy correlated with high expression of MHC, costimulatory and Fas molecules on J558 cells, which was derived from CD40L transgene expression. In addition, transgene CD40L expression could dramatically induce J558 cell apoptosis. Effectively capturing apoptotic bodies by iDCs in vivo could induce DC maturation, prime tumor-specific CTLs and tend to Th1-type immune response. Finally, in vivo depletion experimentation suggested both CD4+ and CD8+ T cells were involved in mediating the antitumor immune responses of combined treatment of AdVCD40L and iDCs, with CD8+ T cells being the major effector. These findings could be beneficial for designing strategies of DCs vaccine and CD40L for anticancer immunotherapy.

Combined alpha tumor necrosis factor gene therapy and engineered dendritic cell vaccine in combating well-established tumors

Although current immunotherapeutic strategies including adenovirus (AdV)‐mediated gene therapy and dendritic cell (DC) vaccine can all stimulate antitumor cytotoxic T lymphocyte (CLT) responses, their therapeutic efficiency has still been limited to generation of prophylactic antitumor immunity against re‐challenge with the parental tumor cells or growth inhibition of small tumors in vivo. However, it is the well‐established tumors in animal models that mimic clinical patients with existing tumor burdens. Alpha tumor necrosis factor (TNF‐α) is a multifunctional and immunoregulatory cytokine that induces antitumor activity and activates immune cells such as DCs and T cells. We hypothesized that a combined immunotherapy including gene therapy and DC vaccine would have some advantages over each modality administered as a monotherapy.