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, 161 (2), 197-208

Langerhans Cells From Women With Cervical Precancerous Lesions Become Functionally Responsive Against Human Papillomavirus After Activation With Stabilized Poly-I:C

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Langerhans Cells From Women With Cervical Precancerous Lesions Become Functionally Responsive Against Human Papillomavirus After Activation With Stabilized Poly-I:C

Diane M Da Silva et al. Clin Immunol.

Abstract

Human papillomavirus (HPV)-mediated suppression of Langerhans cell (LC) function can lead to persistent infection and development of cervical intraepithelial neoplasia (CIN). Women with HPV-induced high-grade CIN2/3 have not mounted an effective immune response against HPV, yet it is unknown if LC-mediated T cell activation from such women is functionally impaired against HPV. We investigated the functional activation of in vitro generated LC and their ability to induce HPV16-specific T cells from CIN2/3 patients after exposure to HPV16 followed by treatment with stabilized Poly-I:C (s-Poly-I:C). LC from patients exposed to HPV16 demonstrated a lack of costimulatory molecule expression, inflammatory cytokine secretion, and chemokine-directed migration. Conversely, s-Poly-I:C caused significant phenotypic and functional activation of HPV16-exposed LC, which resulted in de novo generation of HPV16-specific CD8(+) T cells. Our results highlight that LC of women with a history of persistent HPV infection can present HPV antigens and are capable of inducing an adaptive T cell immune response when given the proper stimulus, suggesting that s-Poly-I:C compounds may be attractive immunomodulators for LC-mediated clearance of persistent HPV infection.

Keywords: HPV16; Human papillomavirus; Immune evasion; Langerhans cells.

Figures

Fig. 1
Fig. 1. MHC and costimulatory molecule upregulation on LC from CIN2/3 patients with Poly-ICR treatment
Immature LC from CIN patients were left untreated or exposed to HPV16 VLP. Subsequently, cells were treated with s-Poly-I:C (10 μg/mL Poly-ICR) for 48h. Controls were left untreated, exposed to HPV16 VLP alone, or treated with s-Poly-I:C alone. (A) Representative flow cytometry data from CIN 02 patient LC untreated or exposed to HPV16 VLP or exposed to HPV16 VLP followed by s-Poly I:C. Expression of indicated surface marker is shown in black histograms. Isotype control staining is shown as gray histogram. (B) Fold increases in LC surface marker expression from CIN2/3 patients (N=10 individual patients) based on geometric mean fluorescence intensity (MFI). Each data point represents an individual patient. Horizontal lines indicate group mean ± 95% confidence interval. **p<0.01, ***p<0.001, ****p<0.0001 (Kruskal-Wallis statistical test following by Dunn’s multiple comparisons test).
Fig. 2
Fig. 2. CCR7 expression and migrate to CCL21 in HPV16-exposed LC treated with Poly-ICR
LC were exposed to HPV16 prior to s-Poly-I:C (10 μg/mL Poly-ICR) treatment as described. (A) CCR7 expression was analyzed by flow cytometry. Data represent mean fold increase in CCR7 expression (±95% confidence interval) relative to untreated LC based on MFI from CIN2/3 patients (N=10 individual patients). (B) In vitro migration assay. LC were analyzed for chemotaxis to CCL21 or medium alone through a transwell insert. Shown is the mean number of LC migrating to CCL21 (black bars) compared to spontaneous migration (white bars) (± SEM) of ten CIN2/3 patients relative to untreated LC. ***p<0.001, ****p<0.0001 compared to untreated LC (Kruskal-Wallis statistical test, Dunn’s multiple comparisons post-test).
Fig. 3
Fig. 3. Production of inflammatory cytokines and chemokines by CIN2/3 patient HPV16-exposed LC treated with Poly-ICR
LC from CIN patients (N=10) were left untreated or exposed to HPV16 prior to treatment with s-Poly-I:C (10 μg/mL Poly-ICR). Cell supernatants were analyzed for a panel of 10 cytokines and chemokines using a Bio-Plex Suspension Array System. Data represent the mean (± SEM) analyte concentration from 10 individual patients. *p<0.05, **p<0.01, ****p<0.0001 compared to untreated LC (one-way ANOVA, Tukey’s post-test).
Fig. 4
Fig. 4. HPV16-exposed LC from CIN2/3 patients treated with Poly-ICLC become phenotypically and functionally active
(A) Poly-ICLC induces upregulation of MHC and costimulatory molecules on LC. LC from a CIN patient were left untreated or exposed to HPV16 prior to treatment with s-Poly-I:C (50 μg/mL Poly-ICLC), then analyzed by flow cytometry. Control cells were left untreated or exposed to HPV16 alone. Data is representative of one patient (CIN 06) out of three patients analyzed. Data shown is the mean fold increase in surface marker expression (± SD) of triplicate replicate values relative to untreated LC based on MFI. (B) Poly-ICLC induces HPV16-exposed LC to express CCR7 as measured via flow cytometry. Data shown for one representative patient out of three is shown as the mean fold increase in CCR7 expression (± SD) of triplicate replicate values relative to untreated LC based on MFI. (C) Poly-ICLC induces HPV16-exposed LC to migrate to chemokine CCL21 in vitro. LC from a CIN patient were exposed to HPV16 prior to Poly-ICLC. LC were analyzed for migration to medium or medium supplemented with CCL21. Shown is the mean (± SD) number of LC migrating to CCL21 compared to spontaneous migration of a representative CIN patient performed in triplicate wells. ***p<0.001 compared to both untreated LC and HPV16-exposed LC (Student’s t test).
Fig. 5
Fig. 5. Poly-ICLC induces gene expression associated with interferon response and innate and adaptive immunity in LC from CIN2/3 patients exposed to HPV
LC were left untreated or exposed to HPV16 prior to treatment with Poly-ICLC (50 μg/mL) for 24 h. Total RNA was isolated and gene expression was analyzed using a PCR gene array profiler for 84 human innate and adaptive immune response genes. Shown is a heat map of expression of genes involved in the interferon response, cytokines and chemokines, antigen presentation, and PAMP receptors and associated adapter molecules that were modulated in unexposed LC or HPV16-exposed LC treated with Poly-ICLC from one representative CIN patient. Untreated LC served as the control group for gene expression analysis after normalization to the housekeeping gene HPRT1.

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