Recurrent respiratory papillomatosis: a complex defect in immune responsiveness to human papillomavirus-6 and -11

Abstract

Recurrent respiratory papillomatosis (RRP) is a rare disease of the larynx caused by infection with human papillomaviruses (HPV) -6 or -11, associated with significant morbidity and on occasion mortality. Here we summarize our current understanding of the permissive adaptive and innate responses made by patients with RRP that support chronic HPV infection and prevent immune clearance of these viruses. Furthermore, we provide new evidence of T(H)2-like polarization in papillomas and blood of patients with RRP, restricted CD4 and CD8 Vbeta repertoires, the effect of HPV-11 early protein E6 on T-cell alloreactivity, enriched Langerhans cell presence in papillomas, and evidence that natural killer cells are dysfunctional in RRP. We review the immunogenetic mechanisms that regulate the dysfunctional responses made by patients with RRP in response to HPV infection of the upper airway. In addition, we are identifying T-cell epitopes on HPV-11 early proteins, in the context of human leukocyte antigen (HLA) class II alleles enriched in RRP that should help generate a therapeutic vaccine. Taken together, RRP is a complex, multigene disease manifesting as a tissue and HPV-specific, immune deficiency that prevents effective clearance and/or control of HPV-6 and -11 infection.

Figure 1. A model of RRP Inhibitory Cycle

This model shows the inhibitory cycle of cells and mediators that are generated by HPV proteins in patients with RRP that are described in this review. Polarized macrophages (AAMΦ)/immature dendritic cells (iDCs), memory T_H2-like T-cells and Tregs, generated from naïve T-cells that express T_H2-like/T-regulatory cytokines and chemokines in response to HPV protein drive this cycle. Memory T_H2-like T-cells express IL-4 and IL-10, and T-regulatory cells express IL-10 and TGFβ in response to HPV proteins are essential in the polarization of resting Mϕs to become AAMϕs that express CCL17, CCL18, and IL-10 which recruit and polarize naïve CD4⁺ T-cells to become Tregs and memory T_H2-like T-cells. This establishes and perpetuates an inhibitory cycle by immunocytes that suppress effective anti-HPV T_H1-like function

Figure 2. RRP Patients produce more IL4 than controls

The percent of resting CD4⁺ T-cells in the blood of RRP patients and controls that constitutively express IL-4 is shown. PBMC from patients with RRP (n=4) and controls (n=2) were studied at day 0 (p<0.05), and some patients (n=2), and controls (n=2) were analyzed at day 14. Samples were processed for intracellular cytokine expression by standard methods.

Figure 3. TILs produce IL4 and IFN-γ, and PBMC from patients express IL-10

A) TILs from a papilloma were for 13 days then stimulated with Phorbol-12-myristate-13-acetate/ionomycin (PMA/I). Distinct populations of CD4⁺ cells were evident, based on intracytoplasmic staining for IL-4 and INF-γ, at a ratio of 2.91 to 1 respectively. B) shows IL-10 intracytoplasmic staining of fresh PBMC obtained from a RRP patient and stimulated with PMA/I. Gating was performed on CD3⁺ cells. The frequency of IL-10 secreting CD4⁺ T-cells in PBMC is 1.19%.

Figure 4. Spectratype of T-cell populations from TILS and PBMC

The TCR Vβ spectratypes of CD4+ T-cells in TILs and PBMC from a representative RRP patient (n=4) are shown. All patients showed oligo- and/or monoclonality in some T-cell families. CD8 data also showed oligo/monoclonality in some T-cell families (data not shown).

Figure 5. E6 inhibits alloreactive T-cell killing

PBMC from a representative patient (n=3) with mild/moderate RRP were exposed to E6 or BSA for 20 hrs. prior to mixing with HLA class I and II mismatched and irradiated PBMC in a MLR at E:T ratio of 25:1. At day 6, patient's T-cells were re-challenged with PKH26 labeled PBMC from the same mismatched stimulator. Target cell death was detected using TO-PRO-3 iodide (TP3)(77, 96). Density plots (upper right quadrant = dead target cells) (upper left = live targets), and the histograms, gated on PKH26⁺ targets, shows a decreased number of dead target cells that incorporated TP3 with E6 exposure (5% killing), while maximum killing without E6 (data not shown) and BSA was 30%.

Figure 6. Langerhans cells are present in papilloma tissues

CD1a⁺ (brown) and S100⁺ (red) cells with Langerhans cell morphology in a laryngeal papilloma from a patient with RRP (B, C, E, F) and the absence of these cells in similarly stained control laryngeal tissue (A, D). Immunohistochemistry was performed on 8 μm sections of a paraffin embedded specimen using the Benchmark XT IHC/ISH Staining Module (Ventana Medical Systems, Tucson, AZ) utilizing a 3,3′diaminobenzidine substrate for CD1a (A, B, C) and Enhanced V-Red substrate for S100 (D, E, F).

Figure 7. NK cells from patients have impaired function compared to controls

PBMC was cultured 4hrs in media alone or with the HLA class I devoid cell line, K562, at an effector:target ratio (E:T) of 5:1 in the presence of FITC-conjugated anti-CD107a and monensin(55). Following incubation, cells were stained with the amine-reactive live/dead fixable violet dead cell stain kit and CD3 APC, CD56 PE, CD14 and CD19 PerCP. Samples were analyzed by flow cytometry. Monocytes, B-cells, and dead cells were excluded from the analysis. Results are shown as the percentage of CD107a expression on CD3^-56⁺ NK cells after subtraction of the spontaneous activity. Data are depicted by box plots (median and 75^th/25^th percentiles), whiskers (90th/10th percentiles); and outliers (outside the 95th and 5th percentiles) are shown. Dotted lines represent arithmetic means.