Search

Supplementary Figure 6. Route-dependency of IL-7-Fc administration on recruitment of T cells in cervicovaginal tissue

Supplementary Figure 4. Comparison of Fc-fragment and IL-7-Fc on accumulation of T cells in cervicovaginal tissue

Supplementary Figure 1. Schematic diagram of Fc-fused IL-7 Supplementary Figure 2. FcRn-mediated transcytosis and transport of Fc-fused IL-7 into serum after intravaginal treatment Supplementary Figure 3. Distinct localization patterns of CD4 and CD8 T cells after intravaginal administration of IL-7-Fc Supplementary Figure 4. Comparison of Fc-fragment and IL-7-Fc on accumulation of T cells in cervicovaginal tissue Supplementary Figure 5. Abrogation of T cell accumulation after IL-7RÃŽ{plus minus} blocking Supplementary Figure 6. Route-dependency of IL-7-Fc administration on recruitment of T cells in cervicovaginal tissue

Supplementary Figure 5. Abrogation of T cell accumulation after IL-7Rα blocking

Supplementary Table 1. Tolerable toxicity after repeated intravaginal administration of IL-7-Fc. Information of detailed materials and methods, and supplementary figure legends are contained.

Supplementary Figure 3. Distinct localization patterns of CD4 and CD8 T cells after intravaginal administration of IL-7-Fc

Supplementary Figure 1. Schematic diagram of Fc-fused IL-7

Supplementary Figure 2. FcRn-mediated transcytosis and transport of Fc-fused IL-7 into serum after intravaginal treatment

Purpose: The induction of tissue-localized virus-specific CD8 T-cell response is essential for the development of an effective therapeutic vaccine against genital diseases, such as cervical cancer and genital herpes. Here, we aimed to elucidate the immunologic role of IL7 in the induction of mucosal cellular immunity.Experimental Design: IL7 was engineered through Fc fusion to enhance mucosal delivery across the genital epithelial barrier. The immunomodulatory role of IL7 was evaluated by monitoring the kinetics of various immune cells and measuring the expression of chemokines and cytokines after intravaginal administration of Fc-fused IL7 (IL7-Fc). The antitumor effects of intramuscular human papillomavirus (HPV) DNA vaccine or topical IL7-Fc alone or in a combinational regimen on mice survival were compared using a orthotopic cervical cancer model.Results: Intravaginal treatment of IL7-Fc, but not native IL7, induces upregulation of chemokines (CXCL10, CCL3, CCL4, and CCL5), cytokines (IFNγ, TNFα, IL6, and IL1β), and an adhesion molecule (VCAM-1) in the genital tract, leading to the recruitment of several leukocytes, including CD4, CD8, γδ T cells, and dendritic cells. Importantly, in this murine cervical cancer model, topical administration of IL7-Fc after intramuscular HPV DNA vaccination increases the number of HPV-specific CD8 T cells in the genital mucosa, but not in the spleen, leading to stronger antitumor activity than the HPV DNA vaccine alone.Conclusions: Our findings provide an important insight into the immunomodulatory role of IL7-Fc via topical application and the design of therapeutic vaccine regimen that induces effective genital–mucosal CD8 T-cell responses. Clin Cancer Res; 22(23); 5898–908. ©2016 AACR.

Clonal cell line-based, multigene-modified, off-the-shelf NK cell therapeutics are emerging as the new frontier of adoptive cellular immunotherapy. Here, we utilized a newly established NK cell line, NK101, as a backbone to derive multifaceted killer cells armored with various antitumor modalities through repeated cycles of genetic modification and clonal selection. First, NK101 cells were transduced with a tricistronic lentiviral vector expressing CD7, CD28, and cytosine deaminase (CD). The resulting cell line demonstrated enhanced cytotoxicity against B7+ tumors and exerted bystander killing effects on neighboring tumor cells upon 5-FC treatment. Second, engineered NK101 cells were again transduced with a bicistronic vector expressing membrane-bound interleukin-15 (mbIL-15) and dominant negative TGFβ type II receptor (DNTβRII). Ectopic expression of mbIL-15 resulted in further augmentation of lytic activities against all tested target cells by inducing upregulation of multiple activating receptors, while that of DNTβRII allowed the cells to maintain heightened cytotoxicity in the presence of TGFβ. Finally, dual-transduced NK101 cells were modified to express chimeric antigen receptors (CARs) targeting either a solid tumor antigen (EpCAM) or a hematologic tumor antigen (FLT3). The final engineered products not only demonstrated antigen-specific killing activities in vitro but also exerted strong tumor-inhibitory effects in preclinical models of metastatic solid tumor and hematologic malignancy. Notably, combined treatment with 5-FC further enhanced antitumor efficacy of engineered NK101 in the solid tumor model. Our results demonstrate successful generation of multigene-modified NK101 cell therapeutics exerting diverse mechanisms of antitumor action – activation receptor-mediated innate killing, antigen-specific killing, and bystander effect-mediated killing.

Inhibition of immune checkpoint proteins like programmed death 1 (PD-1) is a promising therapeutic approach for several cancers, including non-small cell lung cancer (NSCLC). Although PD-1 ligand (PD-L1) expression is used to predict anti-PD-1 therapy responses in NSCLC, its accuracy is relatively less. Therefore, we sought to identify a more accurate predictive blood biomarker for evaluating anti-PD-1 response. We evaluated the frequencies of T cells, B cells, natural killer (NK) cells, polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs), mononuclear myeloid-derived suppressor cells (M-MDSCs), and Lox-1+ PMN-MDSCs in peripheral blood samples of 62 NSCLC patients before and after nivolumab treatment. Correlation of immune-cell population frequencies with treatment response, progression-free survival, and overall survival was also determined. After the first treatment, the median NK cell percentage was significantly higher in responders than in non-responders, while the median Lox-1+ PMN-MDSC percentage showed the opposite trend. NK cell frequencies significantly increased in responders but not in non-responders. NK cell frequency inversely correlated with that of Lox-1+ PMN-MDSCs after the first treatment cycle. The NK cell-to-Lox-1+ PMN-MDSC ratio (NMR) was significantly higher in responders than in non-responders. Patients with NMRs ≥ 5.75 after the first cycle had significantly higher objective response rates and longer progression-free and overall survival than those with NMRs

Background Human natural killer (NK) cell lines serve as an attractive source for adoptive immunotherapy, but NK-92 remains the only cell line being assessed in the clinic. Here, we established a novel NK cell line, NK101, from a patient with extra-nodal natural killer/T-cell lymphoma and examined its phenotypic, genomic and functional characteristics. Methods Single cell suspensions from lymphoma tissue were expanded with anti-NKp46/anti-CD2-coated beads in the presence of IL-2. A continuously growing CD56+ cell clone was selected and designated as NK101. Flow cytometry and RNA sequencing were used to characterize phenotypic and genomic features of NK101. In vitro cytotoxicity and IFN-γ/TNF-α secretion were measured by flow cytometry-based cytotoxicity assay and enzyme-linked immunosorbent assay, respectively, after direct co-culture with tumor cells. Immunomodulatory potential of NK101 was assessed in an indirect co-culture system using conditioned medium. Finally, in vivo antitumor efficacy was evaluated in an immunocompetent, syngeneic 4T1 mammary tumor model. Results NK101 displayed features of CD56dimCD62L+ intermediate stage NK subset with the potential to simultaneously act as a cytokine producer and a cytotoxic effector. Comparative analysis of NK101 and NK-92 revealed that NK101 expressed lower levels of perforin and granzyme B that correlated with weaker cytotoxicity, but produced higher levels of pro-inflammatory cytokines including IFN-γ and TNF-α. Contrarily, NK-92 produced greater amounts of anti-inflammatory cytokines, IL-1 receptor antagonist and IL-10. Genome-wide analysis revealed that genes associated with positive regulation of leukocyte proliferation were overexpressed in NK101, while those with opposite function were highly enriched in NK-92. The consequence of such expressional and functional discrepancies was well-represented in (i) indirect co-culture system where conditioned medium derived from NK101 induced greater proliferation of human peripheral blood mononuclear cells and (ii) immunocompetent 4T1 tumor model where peritumoral injections of NK101 displayed stronger anti-tumor activities by inducing higher tumor-specific immune responses. In a manufacturing context, NK101 not only required shorter recovery time after thawing, but also exhibited faster growth profile than NK-92, yielding more than 200-fold higher cell numbers after 20-day culture. Conclusion NK101 is a unique NK cell line bearing strong immunostimulatory potential and substantial scalability, providing an attractive source for adoptive cancer immunotherapy. Electronic supplementary material The online version of this article (10.1186/s40425-019-0612-2) contains supplementary material, which is available to authorized users.

IgE is central to the development of allergic diseases, and its neutralization alleviates allergic symptoms. However, most of these antibodies are based on IgG1, which is associated with an increased risk of fragment crystallizable-mediated side effects. Moreover, omalizumab, an anti-IgE antibody approved for therapeutic use, has limited benefits for patients with high IgE levels. Here, we assess a fusion protein with extracellular domain of high affinity IgE receptor, FcεRIα, linked to a IgD/IgG4 hybrid Fc domain we term IgETRAP, to reduce the risk of IgG1 Fc-mediated side effects. IgETRAP shows enhanced IgE binding affinity compared to omalizumab. We also see an enhanced therapeutic effect of IgETRAP in food allergy models when combined with Bifidobacterium longum, which results in mast cell number and free IgE levels. The combination of IgETRAP and B. longum may therefore represent a potent treatment for allergic patients with high IgE levels.

Purpose: The induction of tissue-localized virus-specific CD8 T-cell response is essential for the development of an effective therapeutic vaccine against genital diseases, such as cervical cancer and genital herpes. Here, we aimed to elucidate the immunologic role of IL7 in the induction of mucosal cellular immunity. Experimental Design: IL7 was engineered through Fc fusion to enhance mucosal delivery across the genital epithelial barrier. The immunomodulatory role of IL7 was evaluated by monitoring the kinetics of various immune cells and measuring the expression of chemokines and cytokines after intravaginal administration of Fc-fused IL7 (IL7-Fc). The antitumor effects of intramuscular human papillomavirus (HPV) DNA vaccine or topical IL7-Fc alone or in a combinational regimen on mice survival were compared using a orthotopic cervical cancer model. Results: Intravaginal treatment of IL7-Fc, but not native IL7, induces upregulation of chemokines (CXCL10, CCL3, CCL4, and CCL5), cytokines (IFNγ, TNFα, IL6, and IL1β), and an adhesion molecule (VCAM-1) in the genital tract, leading to the recruitment of several leukocytes, including CD4, CD8, γδ T cells, and dendritic cells. Importantly, in this murine cervical cancer model, topical administration of IL7-Fc after intramuscular HPV DNA vaccination increases the number of HPV-specific CD8 T cells in the genital mucosa, but not in the spleen, leading to stronger antitumor activity than the HPV DNA vaccine alone. Conclusions: Our findings provide an important insight into the immunomodulatory role of IL7-Fc via topical application and the design of therapeutic vaccine regimen that induces effective genital–mucosal CD8 T-cell responses. Clin Cancer Res; 22(23); 5898–908. ©2016 AACR.

Genital herpes is one of the most common sexually transmitted infections (STIs), and it is mainly caused by the neurotropic herpes simplex virus (HSV-2). Not only does this infection cause ulcers, but HSV-2 can also stay in a latent state in the nervous system of the host throughout their lifespan. As a result, many people do not know that they harbor this infection. Moreover, HSV-2 serves as a major risk factor for human immunodeficiency virus (HIV) infection and can be transmitted to the fetus. Despite the high risk of infection and adverse effects, attempts at development of an effective vaccine for HSV-2 have not yet been successful. In this study, we developed a DNA vaccine for HSV-2 (SL-V20). This multivalent DNA vaccine effectively reduced the pathological symptoms of infection and induced efficient elimination of the virus in a mouse model. Intramuscular injection of SL-V20 led to induction of an HSV-2-specific T-cell response in the vagina, the major infection site, and in draining lymph organs. Dendritic cells (DCs), especially basic leucine zipper ATF-like transcription factor 3 (Baft3)+ DCs and partially interferon regulatory factor 4 (Irf4)+ DCs, were involved in this T-cell-mediated protective response, while B cells were dispensable for these prophylactic effects. This study demonstrates that SL-V20 offers a novel and effective vaccine against vaginal HSV-2 infection and may be applicable to patients, pending validation in clinical studies.

Objective: Efficient and highly predictive biomarkers reflecting the prognosis of persistent atypical squamous cells of unknown significance(ASCUS) and low grade squamous intraepithelial lesion(LSIL)s are unavailable and need to be developed urgently. We aimed to develop a predictive model for diagnosis of cervical intraepithelial neoplasia(CIN)2+ by analyzing the immunocytochemical expression of the HPV L1 capsid protein in patients with persistent ASCUS and LSIL with a high risk of HPV infection. Methods: Cervical cytology samples comprising (70 ASCUS and 215 LSIL Pap smears) were analyzed. Immunocytochemical identification of the HPV L1 capsid protein in cervical cytology samples was performed. Expression levels of HPV L1 capsid protein in cervical cytology samples were measured, and the correlation between HPV L1 expression and cervical pathologic diagnosis was evaluated. The risk for CIN2+ was calculated using the results of immunocytochemistry and the HPV DNA test. Results: Negative results for HPV L1 immunochemistry test were more frequently observed in CIN2+, and expression of the HPV L1 capsid protein was higher in CIN1 or cervicitis (Fisher's exact test, p

OBJECTIVE Human papillomavirus (HPV) 16 is the most carcinogenic HPV genotype. We investigated if HPV16 L1 capsid protein and E2/E6 ratio, evaluated by cervical cytology, may be used as biomarkers of ≥cervical intraepithelial neoplasia (CIN) 2 lesions. METHODS Cervical specimens were obtained from 226 patients with HPV16 single infection. Using cytology specimen, L1 capsid protein and E2/E6 ratio were detected and the results were compared with those of the conventional histologic analysis of cervical tissues (CIN1-3 and squamous cell carcinoma [SCC]) to evaluate the association. RESULTS The L1 positivity of CIN2/3 was significantly lower than that of normal cervical tissue (p

Adenovirus serotype 5 (Ad5) is one of the most widely used viral vectors and is known to generate potent T cell responses. While many previous studies have characterized Ad5-induced CD8 T cell responses, there is a relative lack of detailed studies that have analyzed CD4 T cells elicited by Ad5 vaccination. Here, we immunized mice with Ad5 vectors encoding lymphocytic choriomeningitis virus (LCMV) glycoprotein (GP) and examined GP-specific CD4 T cell responses elicited by Ad5 vectors and compared them to those induced by an acute LCMV infection. In contrast to LCMV infection, where balanced CD4 T helper 1 (Th1) and T follicular helper (Tfh) responses were induced, Ad5 immunization resulted in a significantly reduced frequency of Th1 cells. CD4 T cells elicited by Ad5 vectors expressed decreased levels of Th1 markers, such as Tim3, SLAM, T-bet, and Ly6C, had smaller amounts of cytotoxic molecules like granzyme B, and produced less interferon gamma than CD4 T cells induced by LCMV infection. This defective CD4 Th1 response appeared to be intrinsic for Ad5 vectors and not a reflection of comparing a nonreplicating vector to a live viral infection, since immunization with a DNA vector expressing LCMV-GP generated efficient CD4 Th1 responses. Analysis at early time points (day 3 or 4) after immunization with Ad5 vectors revealed a defect in the expression of CD25 (interleukin-2 [IL-2] receptor alpha chain) on Ad5-elicited CD4 T cells, and administration of exogenous IL-2 following Ad5 immunization partially restored CD4 Th1 responses. These results suggest that impairment of Th1 commitment after Ad5 immunization could be due to reduced IL-2-mediated signaling. IMPORTANCE During viral infection, generating balanced responses of Th1 and Tfh cells is important to induce effective cell-mediated responses and provide optimal help for antibody responses. In this study, to investigate vaccine-induced CD4 T cell responses, we characterized CD4 T cells after immunization with Ad5 vectors expressing LCMV-GP in mice. Ad5 vectors led to altered effector differentiation of LCMV GP-specific CD4 T cells compared to that during LCMV infection. CD4 T cells following Ad5 immunization exhibited impaired Th1 lineage commitment, generating significantly decreased Th1 responses than those induced by LCMV infection. Our results suggest that suboptimal IL-2 signaling possibly plays a role in reduced Th1 development following Ad5 immunization.

T follicular helper (Tfh) cells are specialized providers of cognate B cell help, which is important in promoting the induction of high-affinity antibody production in germinal centers (GCs). Interleukin-6 (IL-6) and IL-21 have been known to play important roles in Tfh cell differentiation. Here, we demonstrate that IL-7 plays a pivotal role in Tfh generation and GC formation in vivo , as treatment with anti-IL-7 neutralizing antibody markedly impaired the development of Tfh cells and IgG responses. Moreover, codelivery of mouse Fc-fused IL-7 (IL-7-mFc) with a vaccine enhanced the generation of GC B cells as well as Tfh cells but not other lineages of T helper cells, including Th1, Th2, and Th17 cells. Interestingly, a 6-fold-lower dose of an influenza virus vaccine codelivered with Fc-fused IL-7 induced higher antigen-specific and cross-reactive IgG titers than the vaccine alone in both mice and monkeys and led to markedly enhanced protection against heterologous influenza virus challenge in mice. Enhanced generation of Tfh cells by IL-7-mFc treatment was not significantly affected by the neutralization of IL-6 and IL-21, indicating an independent role of IL-7 on Tfh differentiation. Thus, IL-7 holds promise as a critical cytokine for selectively inducing Tfh cell generation and enhancing protective IgG responses. IMPORTANCE Here, we demonstrate for the first time that codelivery of Fc-fused IL-7 significantly increased influenza virus vaccine-induced antibody responses, accompanied by robust expansion of Tfh cells and GC B cells as well as enhanced GC formation. Furthermore, IL-7-mFc induced earlier and cross-reactive IgG responses, leading to striking protection against heterologous influenza virus challenge. These results suggest that Fc-fused IL-7 could be used for inducing strong and cross-protective humoral immunity against highly mutable viruses, such as HIV and hepatitis C virus, as well as influenza viruses.

The inhibitory receptor programmed cell death 1 (PD-1) plays a major role in functional exhaustion of T cells during chronic infections and cancer, and recent clinical data suggest that blockade of the PD-1 pathway is an effective immunotherapy in treating certain cancers. Thus, it is important to define combinatorial approaches that increase the efficacy of PD-1 blockade. To address this issue, we examined the effect of IL-2 and PD-1 ligand 1 (PD-L1) blockade in the mouse model of chronic lymphocytic choriomeningitis virus (LCMV) infection. We found that low-dose IL-2 administration alone enhanced CD8+ T cell responses in chronically infected mice. IL-2 treatment also decreased inhibitory receptor levels on virus-specific CD8+ T cells and increased expression of CD127 and CD44, resulting in a phenotype resembling that of memory T cells. Surprisingly, IL-2 therapy had only a minimal effect on reducing viral load. However, combining IL-2 treatment with blockade of the PD-1 inhibitory pathway had striking synergistic effects in enhancing virus-specific CD8+ T cell responses and decreasing viral load. Interestingly, this reduction in viral load occurred despite increased numbers of Tregs. These results suggest that combined IL-2 therapy and PD-L1 blockade merits consideration as a regimen for treating human chronic infections and cancer.

Adenovirus (Ad) vectors are widely used as experimental vaccines against several infectious diseases, but the magnitude, phenotype, and functionality of CD8 + T cell responses induced by different adenovirus serotypes have not been compared. To address this question, we have analyzed simian immunodeficiency virus Gag-specific CD8 + T cell responses in mice following vaccination with Ad5, Ad26, and Ad35. Our results show that although Ad5 is more immunogenic than Ad26 and Ad35, the phenotype, function, and recall potential of memory CD8 + T cells elicited by these vectors are substantially different. Ad26 and Ad35 vectors generated CD8 + T cells that display the phenotype and function of long-lived memory T cells, whereas Ad5 vector-elicited CD8 + T cells are of a more terminally differentiated phenotype. In addition, hepatic memory CD8 + T cells elicited by Ad26 and Ad35 mounted more robust recall proliferation following secondary challenge than those induced by Ad5. Furthermore, the boosting potential was higher following priming with alternative-serotype Ad vectors than with Ad5 vectors in heterologous prime-boost regimens. Anamnestic CD8 + T cell responses were further enhanced when the duration between priming and boosting was extended from 30 to 60 days. Our results demonstrate that heterologous prime-boost vaccine regimens with alternative-serotype Ad vectors elicited more functional memory CD8 + T cells than any of the regimens containing Ad5. In summary, these results suggest that alternative-serotype Ad vectors will prove useful as candidates for vaccine development against human immunodeficiency virus type 1 and other pathogens and also emphasize the importance of a longer rest period between prime and boost for generating optimal CD8 + T cell immunity.

To design successful vaccines for chronic diseases, an understanding of memory CD8+ T cell responses to persistent antigen re-stimulation is critical. However, most studies comparing memory and naïve cell responses have only been performed in rapidly cleared acute infections. Herein, by comparing the responses of memory and naïve CD8+ T cells to acute and chronic lymphocytic choriomeningitis virus (LCMV) infection, we show that memory cells dominated over naïve cells and were protective when present in sufficient numbers to quickly reduce infection. In contrast, when infection was not rapidly reduced, memory cells were quickly lost, unlike naïve cells. This occurred with both transgenic and endogenous memory CD8+ T cells, and with memory cells initially generated by different vaccines. This loss of memory cells was due to a block in sustaining cell proliferation, selective regulation by the inhibitory receptor 2B4, and increased reliance on CD4+ T cell help. Thus, emphasizing the importance of designing vaccines that elicit effective CD4+ T cell help and rapidly control infection.

Inhibitory receptors play a crucial role in regulating CD8 T-cell function during chronic viral infection. T-cell Ig- and mucin-domain–containing molecule–3 (Tim-3) is well known to negatively regulate T-cell responses, but its role in CD8 T-cell exhaustion during chronic infection in vivo remains unclear. In this study, we document coregulation of CD8 T cell exhaustion by Tim-3 and PD-1 during chronic lymphocytic choriomeningitis virus infection. Whereas Tim-3 was only transiently expressed by CD8 T cells after acute infection, virus-specific CD8 T cells retained high Tim-3 expression throughout chronic infection. The majority (approximately 65% to 80%) of lymphocytic choriomeningitis virus–specific CD8 T cells in lymphoid and nonlymphoid organs coexpressed Tim-3 and PD-1. This coexpression of Tim-3 and PD-1 was associated with more severe CD8 T-cell exhaustion in terms of proliferation and secretion of effector cytokines such as IFN-γ, TNF-α, and IL-2. Interestingly, CD8 T cells expressing both inhibitory receptors also produced the suppressive cytokine IL-10. Most importantly, combined blockade of Tim-3 and PD-1 pathways in vivo synergistically improved CD8 T cell responses and viral control in chronically infected mice. Taken together, our study defines a parameter for determining the severity of CD8 T cell dysfunction and for identifying virus-specific CD8 T cells that produce IL-10, and shows that targeting both PD-1 and Tim-3 is an effective immune strategy for treating chronic viral infections.

Since human papillomavirus (HPV) E6 and E7 are promising tumor antigens, we engineered E6 and E7 antigens to generate an optimal HPV DNA vaccine by codon optimization (Co), fusion of E6 and E7, addition of a tissue plasminogen activator (tpa) signal sequence, addition of CD40 ligand (CD40L) or Fms-like tyrosine kinase-3 ligand (Flt3L). The resulting constructs were investigated in terms of their antitumor activity as well as induction of HPV-specific CD8(+) T cell responses. When E6(Co) and E7(Co) were fused (E67(Co)), CD8(+) T cell responses specific for E6 or E7 antigen decreased, but the preventive antitumor effect rather improved, demonstrating the importance of broad immunity. Interestingly, Flt3L-fused HPV DNA vaccine exhibited stronger E6- and E7-specific CD8(+) T cell responses as well as therapeutic antitumor effect than that of CD40L linked HPV DNA vaccine. Finally, the optimal construct, tFE67(Co), was generated by including tpa signal sequence, Flt3L, fusion of E6 and E7, and codon optimization, which induces 23 and 25 times stronger E6- and E7-specific CD8(+) T cell responses than those of initial E67 fusion construct. In particular, inclusion of electroporation in intramuscular immunization of tFE67(Co) further enhances HPV-specific CD8(+) T cell responses, leading to complete tumor regression in a therapeutic setting. Thus, our results provide valuable insight on effective HPV DNA vaccine design and suggest that tFE67(Co) delivered with electroporation may be a promising therapeutic HPV DNA vaccine against cervical cancer.

Corrigendum: An IL-27/NFIL3 signalling axis drives Tim-3 and IL–10 expression and T-cell dysfunction

Interleukin-12 (IL-12) gene was shown to produce both IL-12 and p40 subunit. The excess production of the p40 subunit as a natural antagonist of IL-12 is a major obstacle of IL-12 gene-based cancer therapy. We previously reported that IL-12N220L gene, which selectively reduces the secretion of the p40 subunit, induces long-lasting stronger type 1 helper T cells (T(H)1) and cytotoxic T lymphocyte (CTL) immunity in hepatitis C virus (HCV) E2 DNA vaccination model and higher protection from challenge with tumor cells expressing E2 than IL-12 in a prophylactic setting. Here, we demonstrated that intratumoral injection of IL-12N220L-expressing adenovirus showed better tumor growth inhibition and higher survival rate than that of IL-12 or granulocyte macrophage-colony stimulating factor (GM-CSF)-expressing adenovirus in a therapeutic setting. In particular, the mice cured by IL-12N220L treatment were protected against intravenous rechallenge of the same tumor cells better than those by IL-12 treatment. In addition, the enhanced antitumor activity of IL-12N220L was confirmed in B16F10 lung metastasis model, which correlated with the frequency of tumor-specific interferon (IFN)-gamma-secreting cells. When tested in CT26/NP tumor that expresses influenza nucleoprotein (NP) as a tumor antigen, IL-12N220L induced stronger NP-specific T(H)1 and CTL responses than IL-12, particularly at a later time point, indicating the generating long-term tumor-specific memory T-cell responses. Moreover, the potent antitumor effects of IL-12N220L were further augmented by combination with chemotherapy using farnesyltransferase inhibitor (FTI), LB42908. Taken together, our results suggest that IL-12N220L is superior to IL-12 in cancer immunotherapy, which can be further enhanced by combination with chemotherapy.

Immune correlates of protection against hepatitis C virus (HCV) infection are not well understood. Here we investigated 2 naive and 6 immunized chimpanzees before and after intravenous challenge, 12 weeks after the last immunization, with 100 50% chimpanzee infectious doses (CID50) of heterologous genotype 1b HCV. Vaccination with recombinant DNA and adenovirus vaccines expressing HCV core, E1E2, and NS3-5 genes induced long-term HCV-specific antibody and T-cell responses and reduced peak viral load about 100 times compared with controls (5.91 ± 0.38 vs. 3.81 ± 0.71 logs, respectively). There was a statistically significant inverse correlation between peak viral loads and envelope glycoprotein 2 (E2)-specific antibody responses at the time of challenge. Interestingly, one vaccinee that had sterilizing immunity against slightly heterologous virus generated the highest level of E2-specific total and neutralizing antibody responses as well as strong NS3/NS5-specific T-cell proliferative responses. The other four vaccinees with low levels of E2-specific antibody had about 44-fold reduced peak viral loads but eventually developed persistent infections. In conclusion, vaccine-induced E2-specific antibody plays an important role in prevention from nonhomologous virus infection and may provide new insight into the development of an effective HCV vaccine. (HEPATOLOGY 2005;42:1429–1436.)

Interleukin-12 (IL-12), consisting of p40 and p35 subunits, produces both p70 heterodimer and free p40. p70 is essential for the induction of T-helper 1 (Th1) and cytotoxic T-cell (CTL) immunity, whereas p40 inhibits p70-mediated function. Here, we found that mutations introduced into N-glycosylation sites (N220 of murine p40 and N222 of human p40) reduced secretion of p40 but not p70. Co-immunization of N220 mutant mIL-12 gene with hepatitis C virus (HCV) E2 DNA significantly enhanced long-term E2-specific CD8+ T-cell response and protection against tumor challenge compared with that of wild type. Our results indicate that the ratio of p70 to p40 is important for generating sustained long-term cell-mediated immunity. Thus, the mutant IL-12 could be utilized for the development of DNA vaccines as an adjuvant for the generation of long-term memory T-cell responses.

Here, we demonstrate that electroporation-enhanced immunization with a rationally designed HPV DNA vaccine (GX-188E), preferentially targeting HPV antigens to dendritic cells, elicits a significant E6/E7-specific IFN-γ-producing T-cell response in all nine cervical intraepithelial neoplasia 3 (CIN3) patients. Importantly, eight out of nine patients exhibit an enhanced polyfunctional HPV-specific CD8 T-cell response as shown by an increase in cytolytic activity, proliferative capacity and secretion of effector molecules. Notably, seven out of nine patients display complete regression of their lesions and viral clearance within 36 weeks of follow up. GX-188E administration does not elicit serious vaccine-associated adverse events at all administered doses. These findings indicate that the magnitude of systemic polyfunctional CD8 T-cell response is the main contributing factor for histological, cytological and virological responses, providing valuable insights into the design of therapeutic vaccines for effectively treating persistent infections and cancers in humans., While several human papilloma virus (HPV) vaccines exist, a highly effective vaccine that mediates regression of HPV-induced tumours is lacking. Here the authors show that a therapeutic DNA vaccine-induced HPV-specific polyfunctional CD8 T cell in 7 out of 9 patients who all exhibited complete regression of lesions and viral clearance.

T cell exhaustion develops under conditions of antigen-persistence caused by infection with various chronic pathogens, such as human immunodeficiency virus (HIV) and mycobacterium tuberculosis (TB), or by the development of cancer. T cell exhaustion is characterized by stepwise and progressive loss of T cell function, which is probably the main reason for the failed immunological control of chronic pathogens and cancers. Recent observations have detailed some of the intrinsic and extrinsic factors that influence the severity of T cell exhaustion. Duration and magnitude of antigenic activation of T cells might be associated with up-regulation of inhibitory receptors, which is a major intrinsic factor of T cell exhaustion. Extrinsic factors might include the production of suppressive cytokines, T cell priming by either non-professional antigen-presenting cells (APCs) or tolerogenic dendritic cells (DCs), and alteration of regulatory T (Treg) cells. Further investigation of the cellular and molecular processes behind the development of T cell exhaustion can reveal therapeutic targets and strategies for the treatment of chronic infections and cancers. Here, we report the properties and the mechanisms of T cell exhaustion in a chronic environment.

Background Current influenza vaccines based on the hemagglutinin protein are strain specific and do not provide good protection against drifted viruses or emergence of new pandemic strains. An influenza vaccine that can confer cross-protection against antigenically different influenza A strains is highly desirable for improving public health. Methodology/Principal Findings To develop a cross protective vaccine, we generated influenza virus-like particles containing the highly conserved M2 protein in a membrane-anchored form (M2 VLPs), and investigated their immunogenicity and breadth of cross protection. Immunization of mice with M2 VLPs induced anti-M2 antibodies binding to virions of various strains, M2 specific T cell responses, and conferred long-lasting cross protection against heterologous and heterosubtypic influenza viruses. M2 immune sera were found to play an important role in providing cross protection against heterosubtypic virus and an antigenically distinct 2009 pandemic H1N1 virus, and depletion of dendritic and macrophage cells abolished this cross protection, providing new insight into cross-protective immune mechanisms. Conclusions/Significance These results suggest that presenting M2 on VLPs in a membrane-anchored form is a promising approach for developing broadly cross protective influenza vaccines.

Programmed cell death-1 (PD-1) is a member of the CD28 superfamily that delivers negative signals upon interaction with its two ligands, PD-L1 or PD-L2. PD-1 and its ligands are broadly expressed and exert a wider range of immunoregulatory roles in T cells activation and tolerance compared with other CD28 members. Subsequent studies show that PD-1-PD-L interaction regulates the induction and maintenance of peripheral tolerance and protect tissues from autoimmune attack. PD-1 and its ligands are also involved in attenuating infectious immunity and tumor immunity, and facilitating chronic infection and tumor progression. The biological significance of PD-1 and its ligand suggests the therapeutic potential of manipulation of PD-1 pathway against various human diseases. In this review, we summarize our current understanding of PD-1 and its ligands ranging from discovery to clinical significance.

Programmed cell death-1 (PD-1) is a member of the CD28 superfamily that delivers negative signals upon interaction with its two ligands, PD-L1 or PD-L2. PD-1 and its ligands are broadly expressed and exert a wider range of immunoregulatory roles in T cells activation and tolerance compared with other CD28 family members. Subsequent studies show that PD-1–PD-L interaction regulates the induction and maintenance of peripheral tolerance and protect tissues from autoimmune attack. PD-1 and its ligands are also involved in attenuating infectious immunity and tumor immunity, and facilitating chronic infection and tumor progression. The biological significance of PD-1 and its ligand suggests the therapeutic potential of manipulation of PD-1 pathway against various human diseases. In this review, we summarize our current understanding of PD-1 and its ligands ranging from discovery to clinical significance.

IL-7 plays a crucial role in the homeostatic proliferation, differentiation and survival of T cells, as well as in the survival and proliferation of precursor B cells. Here, we demonstrated that utilizing nonlytic Fc-fused IL-7 (IL-7-Fc(m)) as a genetic adjuvant significantly enhanced not only CD4(+) but also CD8(+) T-cell responses by E7 DNA immunization, in addition to improving protection against TC-1-induced tumors in comparison to IL-7 alone. Similar results were obtained in OT-1 adoptive transfer experiments with OVA DNA injection, suggesting independence from antigenic nature and experimental conditions. In particular, the increased frequency of CD8(+) T cells was mainly due to enhanced T-cell proliferation in T-cell priming, and not to decreased cellular apoptosis. Interestingly, the enhanced adjuvant effect was not seen in the co-delivery of lytic Fc-fused IL-7 (IL-7-Fc) which increases T-cell apoptosis as well as T-cell proliferation, suggesting that the T-cell proliferative effect may be neutralized by T-cell apoptosis. Thus, our findings suggest that nonlytic Fc, in contrast to lytic Fc, fusion to cytokines may provide an insight in designing a potent genetic adjuvant for inducing CD4(+) and CD8(+) T-cell responses.

IL-12p40 is a natural antagonist which inhibits IL-12- and IL-23-mediated biological activity by blocking the binding of IL-12/23 to their receptors. Recently, IL-12p40 was also shown to have immune-enhancing activity through the activation of macrophages or dendritic cells. In this study, we investigated the effects of IL-12p40 as a genetic adjuvant on immune modulation using recombinant adenoviruses expressing IL-12p40 (rAd/IL-12p40) and OVA (rAd/OVA). Coimmunization of rAd/IL-12p40 at a low dose (1 × 104 PFU) with rAd/OVA resulted in OVA-specific immune enhancement, while a high dose of rAd/IL-12p40 (1 × 108 PFU) caused significant suppression of CD8+ T cell responses. In addition, the enhancement and suppression of OVA-specific CD8+ T cell responses correlated with antitumor activity against E.G7-OVA tumor challenge, which subsequently affected the survival rate. Moreover, the differential CD8+ T cell response by IL-12p40 was still observed in IL-12Rβ2 knockout (IL-12Rβ2KO), but not in IL-12Rβ1 knockout (IL-12Rβ1KO) mice, indicating that IL-12p40 is a cytokine which can modulate Ag-specific T cell responses depending on IL-12Rβ1. Our findings provide a novel insight on the physiological role of IL-12p40, which can be informative in the design of vaccine strategies and therapeutic regimens.

The inhibitory receptor T-cell immunoglobulin and mucin domain-3 (Tim-3) has emerged as a critical regulator of the T-cell dysfunction that develops in chronic viral infections and cancers. However, little is known regarding the signalling pathways that drive Tim-3 expression. Here, we demonstrate that interleukin (IL)-27 induces nuclear factor, interleukin 3 regulated (NFIL3), which promotes permissive chromatin remodelling of the Tim-3 locus and induces Tim-3 expression together with the immunosuppressive cytokine IL-10. We further show that the IL-27/NFIL3 signalling axis is crucial for the induction of Tim-3 in vivo. IL-27-conditioned T helper 1 cells exhibit reduced effector function and are poor mediators of intestinal inflammation. This inhibitory effect is NFIL3 dependent. In contrast, tumour-infiltrating lymphocytes from IL-27R(-/-) mice exhibit reduced NFIL3, less Tim-3 expression and failure to develop dysfunctional phenotype, resulting in better tumour growth control. Thus, our data identify an IL-27/NFIL3 signalling axis as a key regulator of effector T-cell responses via induction of Tim-3, IL-10 and T-cell dysfunction.

IL-23 is a heterodimeric cytokine composed of a p19 subunit and the p40 subunit of IL-12. IL-23 has proinflammatory activity, inducing IL-17 secretion from activated CD4+ T cells and stimulating the proliferation of memory CD4+ T cells. We investigated the pathogenic role of IL-23 in CD4+ T cells in mice lacking the IL-1R antagonist (IL-1Ra−/−), an animal model of spontaneous arthritis. IL-23 was strongly expressed in the inflamed joints of IL-1Ra−/− mice. Recombinant adenovirus expressing mouse IL-23 (rAd/mIL-23) significantly accelerated this joint inflammation and joint destruction. IL-1β further increased the production of IL-23, which induced IL-17 production and OX40 expression in splenic CD4+ T cells of IL-1Ra−/− mice. Blocking IL-23 with anti-p19 Ab abolished the IL-17 production induced by IL-1 in splenocyte cultures. The process of IL-23-induced IL-17 production in CD4+ T cells was mediated via the activation of Jak2, PI3K/Akt, STAT3, and NF-κB, whereas p38 MAPK and AP-1 did not participate in the process. Our data suggest that IL-23 is a link between IL-1 and IL-17. IL-23 seems to be a central proinflammatory cytokine in the pathogenesis of this IL-1Ra−/− model of spontaneous arthritis. Its intracellular signaling pathway could be useful therapeutic targets in the treatment of autoimmune arthritis.

Respiratory syncytial virus (RSV) is a major cause of lower respiratory infection in young children and the elderly. Studies of mice suggest that RSV suppresses the effector activity of CD8 T cells and the development of pulmonary CD8 T cell memory, in which the impaired effector activity could be recovered by in vitro IL-2 treatment. To investigate the effect of in vivo IL-2 expression on RSV immunity, mice were infected with RSV followed by administration of replication-defective adenovirus expressing IL-2. The effector activity of RSV M2-specific CD8 T cells and the development of CD8 T cell memory in the lung was significantly increased by IL-2 expression. Furthermore, the Ab responses against RSV were augmented by IL-2. Interestingly, weight loss and illness caused by RSV challenge were substantially reduced by IL-2 priming, suggesting that the pathogenesis of RSV-related disease could be prevented by IL-2-mediated enhancement of beneficial immune responses. Thus, our results show that IL-2 has potential to be used as a vaccine adjuvant against RSV infection.

Blocking PD-1 signaling, by administering anti-PD-L1 or anti-PD-1 antibodies, has proven to be effective in partially restoring CD8+ T cell function during multiple chronic infection models. However since PD-L1 blockade does not completely rescue CD8+ T cell function, we have focused on finding treatments that synergize with PD-1 blockade. Our lab has previously shown that IL-2 administration can increase the frequency of LCMV-specific CD8+ T cells and decrease viral load. Using a chronic model of lymphocytic choriomeningitis virus (LCMV) we demonstrate that combined IL-2 administration and PD-L1 blockade induces massive increases in LCMV-specific CD8+ T cells and an increased ability to produce inflammatory cytokines to multiple epitopes, including subdominant epitopes, over PD-L1 blockade alone. Furthermore, combined treatment results in greater reduction of viral loads. Importantly, IL-7Rα (CD127), a marker of functional memory cells that is not seen on exhausted cells, even after anti-PD-L1 treatment, was up-regulated on a population of LCMV-specific CD8+ T cells after IL-2 or, even more drastically, after combined treatment. These results indicate that combined IL-2 and PD-1 blockade may prove a potential therapy for increasing CD8+ T cell responses and viral control in human chronic infections.

DNA immunization induces B and T cell responses to various pathogens and tumors. However, these responses are known to be relatively weak and often transient. Thus, novel strategies are necessary for enhancing immune responses induced by DNA immunization. Here, we demonstrated that co-immunization of influenza virus nucleoprotein (NP) gene significantly enhances humoral and cell-mediated responses to codelivered antigens in mice. We also found that NP DNA coimmunization augments in vivo proliferation of adoptively transferred antigen-specific CD4 and CD8 T cells, which enhanced protective immunity against tumor challenge. Our results suggest that NP DNA can serve as a novel genetic adjuvant in cocktail DNA vaccination.

Adenovirus (Ad) vectors have been widely used for many gene therapy applications because of their high transduction ability and broad tropism. However, their utility for cancer gene therapy is limited by their poor transduction into cancer cells lacking the primary receptor, coxsackievirus and adenovirus receptor (CAR). Methods: To achieve CAR-independent gene transfer via Ad, we pretreated Ad with 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) and analyzed their transduction efficiency into cancer cells in vitro and in vivo comparing with the virus alone. Results: Treatment of DOTAP significantly increased adenoviral gene transfer in tumor cells in vitro. Moreover, DOTAP at an optimum dose (10μg/ml) enhanced IL-12 transgene expression by fivefold in tumor, and twofold in serum after intratumoral injection of adenovirus expressing IL-12N220L (Ad/IL-12N220L). In addition, cotreatment of DOTAP decreased tumor growth rate in the Ad/IL-12N220L-transduced tumor model, finally leading to enhanced survival rate. Conclusion: Our results strongly suggest that DOTAP could be of great utility for improving adenovirus-mediated cancer gene therapy.

Human papillomavirus (HPV) infection is responsible for cervical cancer, a common cancer in women. Since HPV infection and cancer development are controlled by the host immune system, immunotherapy against HPV can be helpful in preventing or treating HPV-associated cervical cancer. Two oncoproteins of HPV16, E6 and E7, are promising targets for immunotherapy against cervical cancer, because they are constitutively expressed in cervical cancer. Methods: Since cellular vaccines using B cells as well as dendritic cells offer an efficient approach to cancer immunotherapy, we opted to use B cells. We evaluated the immunogenicity and anti-tumor effects of a B cell vaccine transduced with HPV16 E6/E7-expressing adenovirus. Results: Vaccination with HPV16 E6/E7-transduced B cells induced E6/E7-specific CD8 + T cell-dependent immune responses and generated anti-tumor effects against E6/E7-expressing TC-1 tumor. The anti-tumor effect induced by this B cell vaccine was similar to that elicited by DC vaccine, showing that B cells can be used as an alternative to dendritic cells for cellular vaccines. Conclusion: Thisstudy has shown the feasibility of using B cells as immunogenic APCs and the potential for developing prophylactic and therapeutic vaccines against HPV-associated cervical cancer using a B cell vaccine transduced with adenovirus expressing HPV16 E6/E7.

Cytokine-activated STAT proteins dimerize and bind to high-affinity motifs, and N-terminal domain-mediated oligomerization of dimers allows tetramer formation and binding to low-affinity tandem motifs, but the functions of dimers versus tetramers are unknown. We generated Stat5a-Stat5b double knockin (DKI) N-domain mutant mice in which STAT5 proteins form dimers but not tetramers, identified cytokine-regulated genes whose expression required STAT5 tetramers, and defined dimer versus tetramer consensus motifs. Whereas Stat5-deficient mice exhibited perinatal lethality, DKI mice were viable; thus, STAT5 dimers were sufficient for survival. Nevertheless, STAT5 DKI mice had fewer CD4(+)CD25(+) T cells, NK cells, and CD8(+) T cells, with impaired cytokine-induced and homeostatic proliferation of CD8(+) T cells. Moreover, DKI CD8(+) T cell proliferation after viral infection was diminished and DKI Treg cells did not efficiently control colitis. Thus, tetramerization of STAT5 is critical for cytokine responses and normal immune function, establishing a critical role for STAT5 tetramerization in vivo.