RESUMEN
Advances in high-throughput sequencing have revolutionized the manner with which we can study T cell responses. We describe a woman who received a human papillomavirus (HPV) therapeutic vaccine called PepCan, and experienced complete resolution of her cervical high-grade squamous intraepithelial lesion. By performing bulk T cell receptor (TCR) ß deep sequencing of peripheral blood mononuclear cells before and after 4 vaccinations, 70 putatively vaccine-specific clonotypes were identified for being significantly increased using a beta-binomial model. In order to verify the vaccine-specificity of these clonotypes, T cells with specificity to a region, HPV 16 E6 91-115, previously identified to be vaccine-induced using an interferon-γ enzyme-linked immunospot assay, were sorted and analyzed using single-cell RNA-seq and TCR sequencing. HPV specificity in 60 of the 70 clonotypes identified to be vaccine-specific was demonstrated. TCR ß bulk sequencing of the cervical liquid-based cytology samples and cervical formalin-fixed paraffin-embedded samples before and after 4 vaccinations demonstrated the presence of these HPV-specific T cells in the cervix. Combining traditional and cutting-edge immunomonitoring techniques enabled us to demonstrate expansion of HPV-antigen specific T cells not only in the periphery but also in the cervix. Such an approach should be useful as a novel approach to assess vaccine-specific responses in various anatomical areas.
Asunto(s)
Vacunas contra el Cáncer/uso terapéutico , Papillomavirus Humano 16/inmunología , Linfocitos Infiltrantes de Tumor/efectos de los fármacos , Vacunas contra Papillomavirus/uso terapéutico , Lesiones Intraepiteliales Escamosas/tratamiento farmacológico , Linfocitos T/efectos de los fármacos , Neoplasias del Cuello Uterino/tratamiento farmacológico , Adulto , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Ensayos Clínicos Fase I como Asunto , Femenino , Genes Codificadores de los Receptores de Linfocitos T , Secuenciación de Nucleótidos de Alto Rendimiento , Interacciones Huésped-Patógeno , Humanos , Activación de Linfocitos/efectos de los fármacos , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/virología , Clasificación del Tumor , RNA-Seq , Inducción de Remisión , Lesiones Intraepiteliales Escamosas/inmunología , Lesiones Intraepiteliales Escamosas/patología , Lesiones Intraepiteliales Escamosas/virología , Linfocitos T/inmunología , Linfocitos T/virología , Factores de Tiempo , Resultado del Tratamiento , Neoplasias del Cuello Uterino/inmunología , Neoplasias del Cuello Uterino/patología , Neoplasias del Cuello Uterino/virologíaRESUMEN
Immunotherapy of vulvar high-grade squamous intraepithelial lesion (vHSIL) is investigated as an alternative for surgery, because of high comorbidity and risk of recurrence. Limited evidence exists on the role and composition of the immune microenvironment in current immunotherapeutic approaches for vHSIL. The vHSIL of 29 patients biopsied before treatment with imiquimod were analyzed by two multiplex seven-color immunofluorescence panels to investigate the pre-existing T-cell and myeloid cell composition in relation to treatment response. The samples were scanned with the Vectra multispectral imaging system. Cells were automatically phenotyped and counted with inForm advanced image analysis software. Cell counts and composition were compared to that of vHSIL patients before therapeutic vaccination (n = 29) and to healthy vulva (n = 27). Our data show that the immune microenvironment of complete responders (CR) to imiquimod resembled the coordinated infiltration with type 1 CD4+ and CD8+ T cells and CD14+ inflammatory myeloid cells also found in healthy vulva. However, more CD8+ T cells and FoxP3+ regulatory T cells were present in CR. The lesions of partial responders (PR) lacked such a coordinated response and displayed an impaired influx of CD14+ inflammatory myeloid cells. Importantly, complete responses after imiquimod or therapeutic vaccination showed the same dependency on a pre-existing coordinated type 1 T-cell and CD14+ myeloid cell infiltration. In conclusion, a good clinical outcome after two different forms of immunotherapy for vHSIL is associated with the presence of a primary inflammatory process resulting in the coordinated influx of several types of immune cells which is then amplified.
Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Imiquimod/administración & dosificación , Lesiones Intraepiteliales Escamosas/tratamiento farmacológico , Neoplasias de la Vulva/tratamiento farmacológico , Adyuvantes Inmunológicos/farmacología , Adulto , Anciano , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/metabolismo , Estudios de Casos y Controles , Recuento de Células , Femenino , Humanos , Imiquimod/farmacología , Inmunoterapia , Persona de Mediana Edad , Células Mieloides/efectos de los fármacos , Células Mieloides/metabolismo , Clasificación del Tumor , Lesiones Intraepiteliales Escamosas/inmunología , Lesiones Intraepiteliales Escamosas/patología , Resultado del Tratamiento , Microambiente Tumoral/efectos de los fármacos , Neoplasias de la Vulva/inmunología , Neoplasias de la Vulva/patologíaRESUMEN
OBJECTIVE: To establish the clearance of cervical human papillomavirus (HPV) infection following postoperative immunotherapy with inosine pranobex in women receiving surgical treatment of established high-grade squamous intraepithelial lesion (HSIL) of the uterine cervix. MATERIALS AND METHODS: Over the six-year study period, 32 women with cervical HPV infection following electroconization (loop electrosurgical excision procedure) of the uterine cervix for established HSIL were randomly divided into two groups: I (n = 10) without and II (n = 22) with postoperative inosine pranobex immunotherapy. Follow-up after 24 and 48 months included cervical testing for HPV persistence and after 12, 24, and 48 months with cytology and colposcopy for dysplasia relapse (confirmed histologically). RESULTS: Relapse monitoring in 32 women after 12 months revealed 1 and 0 HSIL positive in groups I and II, respectively; after 24 months an additional 3 patients in each group were positive; and after 48 months an additional 3 and 1 patients were positive in groups I and II, respectively (p < .05). The groups significantly differed (p < .05) with regard to clearing the most common high-risk HPV genotypes (HPV 16 and HPV 56). CONCLUSIONS: Inosine pranobex immunotherapy in HPV-positive patients following cervical conization significantly increased the clearance of viral infection with high-risk genotypes and reduced relapse of HSIL.
Asunto(s)
Conización/métodos , Inmunoterapia/métodos , Infecciones por Papillomavirus/terapia , Lesiones Intraepiteliales Escamosas/terapia , Neoplasias del Cuello Uterino/terapia , Adolescente , Adulto , Femenino , Estudios de Seguimiento , Humanos , Persona de Mediana Edad , Clasificación del Tumor/métodos , Infecciones por Papillomavirus/inmunología , Infecciones por Papillomavirus/patología , Distribución Aleatoria , Lesiones Intraepiteliales Escamosas/inmunología , Lesiones Intraepiteliales Escamosas/patología , Neoplasias del Cuello Uterino/inmunología , Neoplasias del Cuello Uterino/patología , Adulto JovenRESUMEN
Human papillomavirus (HPV) infection is associated with the vast majority of cervical cancer cases as well as with other anogenital cancers. PepCan is an investigational HPV therapeutic vaccine for treating cervical high-grade squamous intraepithelial lesions. The present study was performed to test whether the cervical microbiome influences vaccine responses and to explore host factors as determinants of the cervical microbiome composition in women with biopsy-proven high-grade squamous intraepithelial lesions. In a recently completed Phase I clinical trial of PepCan, histological response rate of 45% (14 of 31 patients), a significant increase in circulating T-helper type 1 cells, and a significant decrease in HPV 16 viral load were reported. DNA, extracted from liquid cytology specimens collected before and after vaccinations, were amplified and then hybridized to a G4 PhyloChip assay to characterize the microbiome. We describe trends that certain bacterial taxa in the cervix may be enriched in non-responders in comparison to responders (Padj = .052 for phylum Caldithrix and Padj = .059 for phylum Nitrospirae). There was no difference in bacterial diversity between the 2 groups. A permutational analysis of variance performed for various demographic and immune parameters showed significant clustering with microbiome beta diversity for race, HPV 16 status, peripheral T-helper type 1 cells, and HLA-B40 (P = .001, .014, .037, and .024, respectively). Further analyses showed significant differences at the empirical Operational Taxonomic Unit level for race and HPV 16 status. As these results are from a small Phase I study, further studies are needed to examine the role of cervical microbiome in response to HPV therapeutic vaccines.