Epigenetic erosion of H4K20me1 induced by inflammation drives aged stem cell ferroptosis.

Aging is associated with a decline in stem cell functionality and number across the organism. In this study, we aimed to further unravel Muscle Stem Cells (MuSCs) aging by assessing how systemic factors influence MuSC fate decisions through long-term epigenetic landscape remodelling. As aging is intricately linked to a pro-inflammatory shift, we studied the epigenetic effects of inflammatory signals in MuSCs and measured decreased H4K20me1 levels. This loss disrupts MuSC quiescence, largely through epigenetic silencing of Notch target genes. In the setting of inflammatory signals or aging, the lack of Kmt5a and the subsequent absence of de novoH4K20me1 culminate in cell death by ferroptosis. Aged MuSCs manifest abnormal iron metabolism and reduced Gpx4 levels, resulting in the accumulation of intracellular iron, increased reactive oxygen species, genomic instability, and lipid peroxidation. We showed that ferroptosis is the predominant mode of cell death in aged MuSCs, with remarkably high levels of lipid peroxidation; a phenomenon we also observed in aged hematopoietic stem cells. Implementing preventative strategies to inhibit systemic inflammation prevented aged MuSC ferroptosis, preserving their numbers and regenerative capabilities. This intervention significantly enhanced aged muscle regeneration and strength recovery and extended both lifespan and healthspan in mice. This study delineates a previously underappreciated fate trajectory for stem cell aging, and offers meaningful insights into the treatment of age-related disorders.

Blood-brain barrier remodeling in an organ-on-a-chip device shows Dkk1 to be a regulator of early metastasis.

Brain metastases are the most lethal progression event, in part because the biological processes underpinning brain metastases are poorly understood. There is a paucity of realistic models of metastasis, as current in vivo murine models are slow to manifest metastasis. We set out to delineate metabolic and secretory modulators of brain metastases by utilizing two models consisting of in vitro microfluidic devices: 1) a blood brain niche (BBN) chip that recapitulates the blood-brain-barrier and niche; and 2) a migration chip that assesses cell migration. We report secretory cues provided by the brain niche that attract metastatic cancer cells to colonize the brain niche region. Astrocytic Dkk-1 is increased in response to brain-seeking breast cancer cells and stimulates cancer cell migration. Brain-metastatic cancer cells under Dkk-1 stimulation increase gene expression of FGF-13 and PLCB1. Further, extracellular Dkk-1 modulates cancer cell migration upon entering the brain niche.

CMV Retinitis with Panretinal Occlusive Vasculitis.

PURPOSE: To report the clinical features of cytomegalovirus (CMV) retinitis with panretinal occlusive vasculitis. METHODS: Retrospective case series. RESULTS: Four eyes in 3 non-HIV patients (male: female = 3:0) were included. Previous medical history included diabetes mellitus (n = 2), age-related macular degeneration (n = 1), and Multiple myeloma under chemotherapy (n = 1). All patients were treated with oral valganciclovir and intravitreal ganciclovir. Slow resolution of retinitis related retinal opacification was noted in all 4 eyes. Two eyes had anti-viral agents discontinued despite the persistent retinitis related opacification and the lesions slowly resolved in the following months. The final decimal visual acuity was equal to or worse than 0.02 in 3 of the 4 eyes. CONCLUSION: In eyes of CMV retinitis with panretinal occlusive vasculitis, rapid resolution of retinitis lesions is an unreliable sign evaluating the therapeutic efficacy of anti-viral agents. Besides, despite treatment of anti-viral agents, deteriorating vascular occlusion may further endanger macular function.

Transit Time Flow Measurement in Robotic Totally Endoscopic Coronary Artery Bypass: What Do the Numbers Mean?

OBJECTIVE: Transit time flow measurement (TTFM) is valuable for assessing intraoperative graft patency in coronary artery bypass surgery (CAB). The significance of competitive native coronary flow on patency, as predicted by percentage of backflow (%BF) on TTFM, is unknown. This study aims to evaluate intraoperative TTFM parameters, and specifically %BF, in predicting graft patency in robotic totally endoscopic CAB (TECAB). METHODS: We reviewed TTFM parameters in 311 patients undergoing robotic off-pump TECAB at our institution between February 2016 and January 2020. Patients with sequential or Y grafts were excluded, leaving 277 patients with a total of 387 isolated end-to-side grafts (248 left internal mammary artery [LIMA], 149 right IMA [RIMA]). Mean graft flow, diastolic flow, pulsatility index, and %BF were measured intraoperatively. Early postoperative angiograms were obtained in 83 patients undergoing percutaneous coronary intervention for hybrid revascularization, with a total of 125 grafts. Angiograms were independently analyzed and separated into 2 groups based on IMA graft patency, which were patent (FitzGibbon A/B) and nonpatent (FitzGibbon O) groups. RESULTS: Early angiographic patency at a median of 31.0 days after surgery showed 123 (97.1%) patent grafts and 3 (2.9%) occluded grafts in both LIMA and RIMA grafts to both left anterior descending (LAD) and non-LAD targets. Mean graft flow was 77.4 ± 41.6 mL/min. There was no difference in mean flow, pulsatility index, or %BF between the patent and occluded grafts. CONCLUSIONS: Excellent intraoperative flow parameters and early angiographic patency can be obtained via robotic, off-pump TECAB. Our data did not demonstrate an association between intraoperative TTFM evidence of competitive native coronary flow and early angiographic graft outcomes.

A model for continued LVAD patient success: A 4-year readmission-free HeartMate 3 postoperative course.

Unplanned readmissions frequently occur following the implantation of a durable left ventricular assist device (LVAD) due to complications such as gastrointestinal bleeding and driveline infection. There is a paucity of literature describing the incidence of unplanned readmission in patients with a HeartMate 3 (HM3) Left Ventricular Assist System. In this report, we present the successful outcome of a patient with an HM3 LVAD who has experienced no unplanned readmissions in the 4-year post-implant phase. To our knowledge, this is the longest readmission-free case after HM3 implantation. A successful patient outcome was enabled by the use of the modular HM3 device, the postoperative prescription of beta-blockers and omega-3, the presence of strong social support, and open communication between the patient's caregivers and the LVAD team. Reducing the instance of unplanned readmission confers clinical benefits to the patient, as well as reducing the cost burden on the patient and the healthcare system.

Discordance between immunofluorescence and immunohistochemistry C4d staining and outcomes following heart transplantation.

BACKGROUND: Capillary deposition of C4d is an important marker of antibody-mediated rejection (AMR) following heart transplantation (HT). There are two immunopathologic assay methods for detecting C4d: frozen-tissue immunofluorescence (IF) and paraffin immunohistochemistry (IHC). The clinical significance of discrepancy between the results of IF and IHC has not been understood. METHODS AND RESULTS: We reviewed 2187 biopsies from 142 HT recipients who had biopsies with assessment of both IF and IHC staining. Among them, 103 (73%) patients had negative IF and IHC C4d staining (Negative Group) and 32 (23%) patients had positive IF but negative IHC staining (Discordant Group). At the time of positive biopsy, 6 (19%) Discordant patients had graft dysfunction, compared to 5 (5%) Negative patients (p = .022). Cumulative incidence of cellular rejection at 1 year was comparable (31% vs. 29%, p = .46); however, cumulative incidence of AMR was significantly higher in the Discordant group (21% vs. 4%, p = .004). Overall 1-year survival was comparable (90% vs. 96%, p = .24); however, freedom from heart failure (HF) was significantly lower in the Discordant group (70% vs. 96%, p < .001). CONCLUSION: The Discordant group showed higher rates of graft dysfunction, AMR and HF admission than the Negative group.

Robotic Repair of Adult Left-Sided Partial Anomalous Pulmonary Venous Connection.

Isolated anomalous drainage of the left pulmonary vein to the left innominate vein is a rare variant of partial anomalous pulmonary venous connection. Here, we describe 2 adult patients with this variant who underwent successful robotic totally endoscopic repair with anastomosis of the pulmonary vein to the left atrial appendage.

Long-Term Outcome of Punctate Inner Choroidopathy or Multifocal Choroiditis with Active Choroidal Neovascularization Managed with Intravitreal Bevacizumab.

Purpose: To evaluate the long-term outcome of active choroidal neovascularization (CNV) in punctate inner choroidopathy (PIC) or multifocal choroiditis (MFC) after intravitreal bevacizumab treatment.Methods: Retrospective study of consecutive patients of PIC/MFC complicated with active CNV. Outcome measures included best-corrected visual acuity (BCVA), total number of intravitreal injections of bevacizumab and recurrence of CNV. Correlation analysis was performed to find the correlation of various clinical factors and final BCVA.Results: There were 23 eyes in 22 patients with a mean age of 33.22 years included in this study. The mean duration of follow-up was 6.48 years. Improvement of BCVA was noted through the first 3 years and at the final follow-up. BCVA at 1, 6, 12 months and recurrence of CNV were correlated with final BCVA.Conclusion: Most patients of PIC/MFC complicated with CNV managed with intravitreal bevacizumab had improved BCVA over 4 years.

Sympathomimetic Effects of Acute E-Cigarette Use: Role of Nicotine and Non-Nicotine Constituents.

BACKGROUND: Chronic electronic (e) cigarette users have increased resting cardiac sympathetic nerve activity and increased susceptibility to oxidative stress. The purpose of the present study is to determine the role of nicotine versus non-nicotine constituents in e-cigarette emissions in causing these pathologies in otherwise healthy humans. METHODS AND RESULTS: Thirty-three healthy volunteers who were not current e-cigarette or tobacco cigarette smokers were studied. On different days, each participant used an e-cigarette with nicotine, an e-cigarette without nicotine, or a sham control. Cardiac sympathetic nerve activity was determined by heart rate variability, and susceptibility to oxidative stress was determined by plasma paraoxonase activity. Following exposure to the e-cigarette with nicotine, but not to the e-cigarette without nicotine or the sham control, there was a significant and marked shift in cardiac sympathovagal balance towards sympathetic predominance. The decrease in high-frequency component and the increases in the low-frequency component and the low-frequency to high-frequency ratio were significantly greater following exposure to the e-cigarette with nicotine compared with exposure to the e-cigarette without nicotine or to sham control. Oxidative stress, as estimated by plasma paraoxonase, did not increase following any of the 3 exposures. CONCLUSIONS: The acute sympathomimetic effect of e-cigarettes is attributable to the inhaled nicotine, not to non-nicotine constituents in e-cigarette aerosol, recapitulating the same heart rate variability pattern associated with increased cardiac risk in multiple populations with and without known cardiac disease. Evidence of oxidative stress, as estimated by plasma paraoxonase activity, was not uncovered following acute e-cigarette exposure.

Consumption of energy beverage is associated with attenuation of arterial endothelial flow-mediated dilatation.

AIM: To investigate whether consumption of an energy drink will acutely impair endothelial function in young healthy adults. METHODS: Energy drinks are being consumed more and more worldwide, and have been associated with some deaths in adolescents and young adults, especially when consumed while exercising. After fasting and not smoking for at least 8 h prior, eleven medical students (9 males) received an electrocardiogram, blood pressure and pulse check, and underwent baseline testing (BL) of endothelial function using the technique of endothelium-dependent flow mediated dilatation (FMD) with high-resolution ultrasound (according to recommended guidelines of the University of Wisconsin Atherosclerosis Imaging Research Program Core Laboratory). The subjects then drank an energy beverage (EB), a 24-oz can of Monster Energy, and the above was repeated at 90 min after consumption. The relative FMD (%) was calculated as the ratio between the average post-cuff release and the baseline diameter. Each image was checked for quality control, and each artery diameter was measured from the media to media points by two experts, 3 measurements at the QRS complex, repeated on 3 separate beats, and then all were averaged. RESULTS: Subjects characteristics averages (given with standard deviations) include: Age 24.5 ± 1.5 years, sex 9 male and 2 female, weight 71.0 ± 9.1 kg, height 176.4 ± 6.0 cm, BMI 22.8 ± 2.7 kg/m2. The hemodynamics were as follows, BL vs EB group respectively (mean ± SD): Heart rate 65.2 ± 11.3 vs 68.2 ± 11.8 beats per minute, systolic blood pressure 114.0 ± 10.4 mmHg vs 114.1 ± 10.4 mmHg, diastolic blood pressure 68.8 ± 9.3 mmHg vs 70.6 ± 7.1 mmHg; all were not significantly different. However after drinking the EB, a significantly attenuated peak FMD response was measured (mean ± SD): BL group 5.9% ± 4.6% vs EB group 1.9% ± 2.1%; P = 0.03). Given the increased consumption of energy beverages associated with exercise in young adults, more research is needed. CONCLUSION: Energy beverage consumption has a negative impact on arterial endothelial function in young healthy adults.

RPN13/ADRM1 inhibitor reverses immunosuppression by myeloid-derived suppressor cells.

Myeloid-derived-suppressor cells (MDSCs) are key mediators of immune suppression in the ovarian tumor microenvironment. Modulation of MDSC function to relieve immunosuppression may enhance the immunologic clearance of tumors. The bis-benzylidine piperidone RA190 binds to the ubiquitin receptor RPN13/ADRM1 on the 19S regulatory particle of the proteasome and directly kills ovarian cancer cells by triggering proteotoxic stress. Here we examine the effect of RA190 treatment on the immunosuppression induced by MDSCs in the tumor microenvironment, specifically on the immunosuppression induced by MDSCs. We show that RA190 reduces the expression of Stat3 and the levels of key immunosuppressive enzymes and cytokines arginase, iNOS, and IL-10 in MDSCs, while boosting expression of the immunostimulatory cytokine IL-12. Furthermore, we show that the RA190-treated MDSCs lost their capacity to suppress CD8+ T cell function. Finally, we show that RA190 treatment of mice bearing syngeneic ovarian tumor elicits potent CD8+ T cell antitumor immune responses and improves tumor control and survival. These data suggest the potential of RA190 for ovarian cancer treatment by both direct killing of tumor cells via proteasome inhibition and relief of MDSC-mediated suppression of CD8 T cell-dependent antitumor immunity elicited by the apoptotic tumor cells.

Buccal injection of synthetic HPV long peptide vaccine induces local and systemic antigen-specific CD8+ T-cell immune responses and antitumor effects without adjuvant.

BACKGROUND: Human Papillomavirus is responsible for over 99 % of cervical cancers and is associated with cancers of the head and neck. The currently available prophylactic vaccines against HPV do not generate therapeutic effects against established HPV infections and associated lesions and disease. Thus, the need for a therapeutic vaccine capable of treating HPV-induced malignancies persists. Synthetic long peptides vaccination is a popular antigen delivery method because of its safety, stability, production feasibility, and its need to be processed by professional antigen presenting cells before it can be presented to cytotoxic CD8+ T lymphocytes. Cancers in the buccal mucosa have been shown to elicit cancer-related inflammations that are capable of recruiting inflammatory and immune cells to generate antitumor effects. In the current study, we evaluated the therapeutic potential of synthetic HPV long peptide vaccination in the absence of adjuvant in the TC-1 buccal tumor model. RESULT: We show that intratumoral vaccination with E7 long peptide alone effectively controls buccal TC-1 tumors in mice. Furthermore, we observed an increase in systemic as well as local E7-specific CD8+ T cells in buccal tumor-bearing mice following the vaccination. Finally, we show that induction of immune responses against buccal tumors by intratumoral E7 long peptide vaccination is independent of CD4+ T cells, and that the phenomenon may be related to the unique environment associated with mucosal tissues. CONCLUSION: Our results suggest the possibility for clinical translation of the administration of adjuvant free therapeutic long peptide vaccine as a potentially effective and safe strategy for mucosal HPV-associated tumor treatment.

Optimization of heterologous DNA-prime, protein boost regimens and site of vaccination to enhance therapeutic immunity against human papillomavirus-associated disease.

BACKGROUND: Human papillomavirus (HPV) has been identified as the primary etiologic factor of cervical cancer as well as subsets of anogenital and oropharyngeal cancers. The two HPV viral oncoproteins, E6 and E7, are uniquely and consistently expressed in all HPV infected cells and are therefore promising targets for therapeutic vaccination. Both recombinant naked DNA and protein-based HPV vaccines have been demonstrated to elicit HPV-specific CD8+ T cell responses that provide therapeutic effects against HPV-associated tumor models. Here we examine the immunogenicity in a preclinical model of priming with HPV DNA vaccine followed by boosting with filterable aggregates of HPV 16 L2E6E7 fusion protein (TA-CIN). RESULTS: We observed that priming twice with an HPV DNA vaccine followed by a single TA-CIN booster immunization generated the strongest antigen-specific CD8+ T cell response compared to other prime-boost combinations tested in C57BL/6 mice, whether naïve or bearing the HPV16 E6/E7 transformed syngeneic tumor model, TC-1. We showed that the magnitude of antigen-specific CD8+ T cell response generated by the DNA vaccine prime, TA-CIN protein vaccine boost combinatorial strategy is dependent on the dose of TA-CIN protein vaccine. In addition, we found that a single booster immunization comprising intradermal or intramuscular administration of TA-CIN after priming twice with an HPV DNA vaccine generated a comparable boost to E7-specific CD8+ T cell responses. We also demonstrated that the immune responses elicited by the DNA vaccine prime, TA-CIN protein vaccine boost strategy translate into potent prophylactic and therapeutic antitumor effects. Finally, as seen for repeat TA-CIN protein vaccination, we showed that the heterologous DNA prime and protein boost vaccination strategy is well tolerated by mice. CONCLUSIONS: Our results provide rationale for future clinical testing of HPV DNA vaccine prime, TA-CIN protein vaccine boost immunization regimen for the control of HPV-associated diseases.

Current state in the development of candidate therapeutic HPV vaccines.

The identification of human papillomavirus (HPV) as an etiological factor for HPV-associated malignancies creates the opportunity to control these cancers through vaccination. Currently, available preventive HPV vaccines have not yet demonstrated strong evidences for therapeutic effects against established HPV infections and lesions. Furthermore, HPV infections remain extremely common. Thus, there is urgent need for therapeutic vaccines to treat existing HPV infections and HPV-associated diseases. Therapeutic vaccines differ from preventive vaccines in that they are aimed at generating cell-mediated immunity rather than neutralizing antibodies. The HPV-encoded early proteins, especially oncoproteins E6 and E7, form ideal targets for therapeutic HPV vaccines since they are consistently expressed in HPV-associated malignancies and precancerous lesions, playing crucial roles in the generation and maintenance of HPV-associated disease. Our review will cover various therapeutic vaccines in development for the treatment of HPV-associated lesions and cancers. Furthermore, we review strategies to enhance vaccine efficacy and the latest clinical trials on therapeutic HPV vaccines.

Identification of the murine H-2D(b) and human HLA-A*0201 MHC class I-restricted HPV6 E7-specific cytotoxic T lymphocyte epitopes.

Recurrent respiratory papillomatosis is caused by human papillomavirus (HPV) infection, most commonly types 6 (HPV-6) and 11 (HPV-11). Due to failed host immune responses, HPV is unable to be cleared from the host, resulting in recurrent growth of HPV-related lesions that can obstruct the lumen of the airway within the upper aerodigestive tract. In our murine model, the HPV-6b and HPV-11 E7 antigens are not innately immunogenic. In order to enhance the host immune responses against the HPV E7 antigen, we linked calreticulin (CRT) to HPV-6b E7 and found that vaccinating C57BL/6 mice with the HPV-6b CRT/E7 DNA vaccine is able to induce a CD8+ T cell response that recognizes an H-2D(b)-restricted E7aa21-29 epitope. Additionally, vaccination of HLA-A*0201 transgenic mice with HPV-6b CRT/E7 DNA generated a CD8+ T cell response against the E7aa82-90 epitope that was not observed in the wild-type C57BL/6 mice, indicating this T cell response is restricted to HLA-A*0201. In vivo cytotoxic T cell killing assays demonstrated that the vaccine-induced CD8+ T cells are able to efficiently kill target cells. Interestingly, the H-2D(b)-restricted E7aa21-29 sequence and the HLA-A*0201-restricted E7aa82-90 sequence are conserved between HPV-6b and HPV-11 and may represent shared immunogenic epitopes. The identification of the HPV-6b/HPV-11 CD8+ T cell epitopes facilitates the evaluation of various immunomodulatory strategies in preclinical models. More importantly, the identified HLA-A*0201-restricted T cell epitope may serve as a peptide vaccination strategy, as well as facilitate the monitoring of vaccine-induced HPV-specific immunologic responses in future human clinical trials.

Local HPV Recombinant Vaccinia Boost Following Priming with an HPV DNA Vaccine Enhances Local HPV-Specific CD8+ T-cell-Mediated Tumor Control in the Genital Tract.

PURPOSE: Two viral oncoproteins, E6 and E7, are expressed in all human papillomavirus (HPV)-infected cells, from initial infection in the genital tract to metastatic cervical cancer. Intramuscular vaccination of women with high-grade cervical intraepithelial neoplasia (CIN2/3) twice with a naked DNA vaccine, pNGVL4a-sig/E7(detox)/HSP70, and a single boost with HPVE6/E7 recombinant vaccinia vaccine (TA-HPV) elicited systemic HPV-specific CD8 T-cell responses that could traffic to the lesion and was associated with regression in some patients (NCT00788164). EXPERIMENTAL DESIGN: Here, we examine whether alteration of this vaccination regimen by administration of TA-HPV vaccination in the cervicovaginal tract, rather than intramuscular (IM) delivery, can more effectively recruit antigen-specific T cells in an orthotopic syngeneic mouse model of HPV16(+) cervical cancer (TC-1 luc). RESULTS: We found that pNGVL4a-sig/E7(detox)/HSP70 vaccination followed by cervicovaginal vaccination with TA-HPV increased accumulation of total and E7-specific CD8(+) T cells in the cervicovaginal tract and better controlled E7-expressing cervicovaginal TC-1 luc tumor than IM administration of TA-HPV. Furthermore, the E7-specific CD8(+) T cells in the cervicovaginal tract generated through the cervicovaginal route of vaccination expressed the α4ß7 integrin and CCR9, which are necessary for the homing of the E7-specific CD8(+) T cells to the cervicovaginal tract. Finally, we show that cervicovaginal vaccination with TA-HPV can induce potent local HPV-16 E7 antigen-specific CD8(+) T-cell immune responses regardless of whether an HPV DNA vaccine priming vaccination was administered IM or within the cervicovaginal tract. CONCLUSIONS: Our results support future clinical translation using cervicovaginal TA-HPV vaccination.

Pancreatic adenocarcinoma upregulated factor serves as adjuvant by activating dendritic cells through stimulation of TLR4.

Dendritic cell (DC) based cancer vaccines represent a promising immunotherapeutic strategy against cancer. To enhance the modest immunogenicity of DC vaccines, various adjuvants are often incorporated. Particularly, most of the common adjuvants are derived from bacteria. In the current study, we evaluate the use of a human pancreatic cancer derived protein, pancreatic adenocarcinoma upregulated factor (PAUF), as a novel DC vaccine adjuvant. We show that PAUF can induce activation and maturation of DCs and activate NFkB by stimulating the Toll-like receptor signaling pathway. Furthermore, vaccination with PAUF treated DCs pulsed with E7 or OVA peptides leads to generation of E7 or OVA-specific CD8+ T cells and memory T cells, which correlate with long term tumor protection and antitumor effects against TC-1 and EG.7 tumors in mice. Finally, we demonstrated that PAUF mediated DC activation and immune stimulation are dependent on TLR4. Our data provides evidence supporting PAUF as a promising adjuvant for DC based therapies, which can be applied in conjunction with other cancer therapies. Most importantly, our results serve as a reference for future investigation of human based adjuvants.

Co-administration with DNA encoding papillomavirus capsid proteins enhances the antitumor effects generated by therapeutic HPV DNA vaccination.

BACKGROUND: DNA vaccines have emerged as attractive candidates for the control of human papillomavirus (HPV)-associated malignancies. However, DNA vaccines suffer from limited immunogenicity and thus strategies to enhance DNA vaccine potency are needed. We have previously demonstrated that for DNA vaccines encoding HPV-16 E7 antigen (CRT/E7) linkage with calreticulin (CRT) linked enhances both the E7-specific CD8(+) T cell immune responses and antitumor effects against E7-expressing tumors. In the current study, we aim to introduce an approach to elicit potent CD4(+) T cell help for the enhancement of antigen-specific CD8(+) T cell immune responses generated by CRT/E7 DNA vaccination by using co-administration of a DNA vector expressing papillomavirus major and minor capsid antigens, L1 and L2. RESULT: We showed that co-administration of vectors containing codon-optimized bovine papillomavirus type 1 (BPV-1) L1 and L2 in combination with DNA vaccines could elicit enhanced antigen-specific CD8(+) in both CRT/E7 and ovalbumin (OVA) antigenic systems. We also demonstrated that co-administration of vectors expressing BPV-1 L1 and/or L2 DNA with CRT/E7 DNA led to the generation of L1/L2-specific CD4(+) T cell immune responses and L1-specific neutralizing antibodies. Furthermore, we showed that co-administration with DNA encoding BPV1 L1 significantly enhances the therapeutic antitumor effects generated by CRT/E7 DNA vaccination. In addition, the observed enhancement of CD8(+) T cell immune responses by DNA encoding L1 and L2 was also found to extend to HPV-16 L1/L2 system. CONCLUSION: Our strategy elicits both potent neutralizing antibody and therapeutic responses and may potentially be extended to other antigenic systems beyond papillomavirus for the control of infection and/or cancer.

Local administration of granulocyte macrophage colony-stimulating factor induces local accumulation of dendritic cells and antigen-specific CD8+ T cells and enhances dendritic cell cross-presentation.

Immunotherapy has emerged as a promising treatment strategy for the control of HPV-associated malignancies. Various therapeutic HPV vaccines have elicited potent antigen-specific CD8+ T cell mediated antitumor immune responses in preclinical models and are currently being tested in several clinical trials. Recent evidence indicates the importance of local immune activation, and higher number of immune cells in the site of lesion correlates with positive prognosis. Granulocyte macrophage colony-stimulating factor (GMCSF) has been reported to posses the ability to induce migration of antigen presentation cells and CD8+ T cells. Therefore, in the current study, we employ a combination of systemic therapeutic HPV DNA vaccination with local GMCSF application in the TC-1 tumor model. We show that intramuscular vaccination with CRT/E7 DNA followed by GMCSF intravaginal administration effectively controls cervicovaginal TC-1 tumors in mice. Furthermore, we observe an increase in the accumulation of E7-specific CD8+ T cells and dendritic cells in vaginal tumors following the combination treatment. In addition, we show that GMCSF induces activation and maturation in dendritic cells and promotes antigen cross-presentation. Our results support the clinical translation of the combination treatment of systemic therapeutic vaccination followed by local GMCSF administration as an effective strategy for tumor treatment.