Immune Dysregulation in Patients Persistently Infected with Human Papillomaviruses 6 and 11.

Human Papillomaviruses (HPVs) 6 and 11 are part of a large family of small DNA viruses, some of which are commensal. Although much of the population can contain or clear infection with these viruses, there is a subset of individuals who develop persistent infection that can cause significant morbidity and on occasion mortality. Depending on the site of infection, patients chronically infected with these viruses develop either recurrent, and on occasion, severe genital warts or recurrent respiratory papillomas that can obstruct the upper airway. The HPV-induced diseases described are likely the result of a complex and localized immune suppressive milieu that is characteristic of patients with persistent HPV infection. We review data that documents impaired Langerhans cell responses and maturation, describes the polarized adaptive T-cell immune responses made to these viruses, and the expression of class select II MHC and KIR genes that associate with severe HPV6 and 11 induced disease. Finally, we review evidence that documents the polarization of functional TH2 and T-regulatory T-cells in tissues persistently infected with HPV6 and 11, and we review evidence that there is suppression of natural killer cell function. Together, these altered innate and adaptive immune responses contribute to the cellular and humoral microenvironment that supports HPV 6 and 11-induced disease.

Decreased Langerhans cell responses to IL-36γ: altered innate immunity in patients with recurrent respiratory papillomatosis.

Recurrent respiratory papillomatosis (RRP) is a rare, chronic disease caused by human papillomaviruses (HPVs) types 6 and 11 that is characterized by the polarization of adaptive immune responses that support persistent HPV infection. Respiratory papillomas express elevated mRNA levels of IL-36γ, a proinflammatory cytokine in comparison to autologous clinically normal laryngeal tissues; however there is no evidence of inflammation in these lesions. Consistent with this, respiratory papillomas do not contain TH1-like CD4(+) T-cells or cytotoxic CD8(+) T-cells, but instead contain a predominance of TH2-like and T regulatory cells (Tregs). In addition, papillomas also are infiltrated with immature Langerhans cells (iLCs). In this study, we show that papilloma cells express IL-36γ protein, and that human keratinocytes transduced with HPV11 have reduced IL-36γ secretion. We now provide the first evidence that peripheral blood-derived iLCs respond to IL-36γ by expressing inflammatory cytokines and chemokines. When stimulated with IL-36γ, iLCs from patients with RRP had lower expression levels of the TH2-like chemokine CCL-20 as compared with controls. Patients' iLCs also had decreased steady state levels of CCL-1, which is a proinflammatory chemokine. Moreover, CCL-1 levels in iLCs inversely correlated with the severity of RRP. The combined decrease of TH1- and a TH2-like chemokines by iLCs from patients could have consequences in the priming of IFN-γ expression by CD8(+) T-cells. Taken together, our results suggest that, in RRP, there is a defect in the proinflammatory innate immune responses made by iLCs in response to IL-36γ. The consequence of this defect may lead to persistent HPV infection by failing to support an effective HPV-specific, TH1-like and/or Tc1-like adaptive response, thus resulting in the predominant TH2-like and/or Treg micromilieu present in papillomas.

Innate immune recognition of molds and homology to the inner ear protein, cochlin, in patients with autoimmune inner ear disease.

Autoimmune Inner Ear Disease (AIED) is characterized by bilateral, fluctuating sensorineural hearing loss with periods of hearing decline triggered by unknown stimuli. Here we examined whether an environmental exposure to mold in these AIED patients is sufficient to generate a pro-inflammatory response that may, in part, explain periods of acute exacerbation of disease. We hypothesized that molds may stimulate an aberrant immune response in these patients as both several Aspergillus species and penecillium share homology with the LCCL domain of the inner ear protein, cochlin. We showed the presence of higher levels of anti-mold IgG in plasma of AIED patients at dilution of 1:256 (p = 0.032) and anti-cochlin IgG 1:256 (p = 0.0094 and at 1:512 p = 0.024) as compared with controls. Exposure of peripheral blood mononuclear cells (PBMC) of AIED patients to mold resulted in an up-regulation of IL-1ß mRNA expression, enhanced IL-1ß and IL-6 secretion, and generation of IL-17 expressing cells in mold-sensitive AIED patients, suggesting mold acts as a PAMP in a subset of these patients. Naïve B cells secreted IgM when stimulated with conditioned supernatant from AIED patients' monocytes treated with mold extract. In conclusion, the present studies indicate that fungal exposure can trigger autoimmunity in a subset of susceptible AIED patients.

Immune suppression in premalignant respiratory papillomas: enriched functional CD4+Foxp3+ regulatory T cells and PD-1/PD-L1/L2 expression.

PURPOSE: Respiratory papillomas, caused by human papillomaviruses types 6 and 11 (HPV6/11), are premalignant lesions with potential for malignant conversion. The cytokine and chemokine micromilieu of papillomas is T(H)2-like with a marked absence of IFN-γ expression. To illuminate why patients with recurrent respiratory papillomatosis (RRP) fail to effectively control their disease, we further investigated the suppressive cellular microenvironment in papillomas. EXPERIMENTAL DESIGN: CD4(+)CD25(+)CD127(low/-)Foxp3(+) regulatory T cells (Treg) and CD4(+)CD25(-)CD127(low/-)Foxp3(-) T cells within papillomas were characterized and isolated. Their suppressor function was measured by inhibition of peripheral blood mononuclear cell (PBMC) proliferation. Expression of PD-1, CD69, and Helios was identified on these T cells. PD-L1, PD-L2, CCL17, and CCL22 mRNA was also identified in papillomas by quantitative PCR. RESULTS: Functional Tregs were markedly enriched in papillomas and strongly inhibited anti-CD3 and anti-CD28 antibody activated PBMC proliferation. The natural Treg marker Helios was reduced on Tregs from papillomas, indicating that the majority of Tregs in papillomas are adaptive. The majority of the papilloma-derived CD4(+) T cells expressed the CD4(+)CD25(-)CD127(low/-)Foxp3(-)PD1(+)CD69(+) phenotype and failed to suppress PBMC proliferation, suggesting that they are chronically activated and exhausted. The Treg-attracting chemokine CCL22 was equally expressed by all laryngeal tissues examined. However, CCL17 was robustly expressed by papillomas compared with unaffected laryngeal tissues from RRP patients and individuals without RRP. PD-L1 was elevated in papillomas compared with control laryngeal tissues. CONCLUSIONS: Papilloma CD4(+) T cells are enriched with functional Tregs, and the adaptive Helios(-) Treg fraction was increased within the T(H)2-like papilloma micromilieu. CD4(+)CD25(-)CD127(low/-)Foxp3(-) T-cells failed to suppress PBMC proliferation and may be exhausted. The PD-1/PDL-1 pathway may represent an additional immunosuppressive mechanism that contributes to defective HPV6/11 clearance in RRP.

Papillomavirus-specific CD4+ T cells exhibit reduced STAT-5 signaling and altered cytokine profiles in patients with recurrent respiratory papillomatosis.

Recurrent respiratory papillomatosis (RRP) is caused by human papillomavirus type 6 (HPV-6) or HPV-11. Specific HLA-DR haplotypes DRB1*01:02 and DRB1*03:01 are associated with the development of RRP, disease severity, and Th2-like responses to HPV early proteins. Th1-like responses to HPV proteins have been shown to be protective in animal models. Therefore, we investigated the hypothesis that RRP patients have dysfunctional Th1-like, HPV-specific T cell responses. Using MHC class II tetramers, we identified immunogenic peptides within HPV-11 early proteins. Two distinct peptides (E6(113-132) and E2(1-20)) contained DRB1*01:02- or DRB1*03:01-restricted epitopes, respectively. An additional peptide (E2(281-300)) contained an epitope presented by both alleles. Peptide binding, tetramer, and proliferation assays identified minimal epitopes within these peptides. These epitopes elicited E2/E6-specific CD4(+) T cell responses in RRP patients and healthy control subjects, allowing the isolation of HPV-specific T cell lines using tetramers. The cytokine profiles and STAT signaling of these tetramer-positive T cells were measured to compare the polarization and responsiveness of HPV-specific T cells from patients with RRP and healthy subjects. HPV-specific IFN-γ secretion was substantially lower in T cells from RRP patients. HPV-specific IL-13 secretion was seen at modest levels in T cells from RRP patients and was absent in T cells from healthy control subjects. HPV-specific T cells from RRP patients exhibited reduced STAT-5 phosphorylation and reduced IL-2 secretion, suggesting anergy. Levels of STAT-5 phosphorylation and IFN-γ secretion could be improved through addition of IL-2 to HPV-specific T cell lines from RRP patients. Therapeutic vaccination or interventions aimed at restoring Th1-like cytokine responses to HPV proteins and reversing anergy could improve clinical outcomes for RRP patients.

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

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.

Activating killer cell immunoglobulin-like receptors 3DS1 and 2DS1 protect against developing the severe form of recurrent respiratory papillomatosis.

The polymorphic killer cell immunoglobulin-like receptors (KIR) control natural killer (NK) cell response against viral infection and tumor transformation. Here we investigated if select KIR genes are associated with recurrent respiratory papillomatosis (RRP), a rare disease of the larynx and upper airway caused by human papillomaviruses (HPV)-6/11. DNA from 66 RRP patients and 195 healthy controls were characterized for KIR and HLA gene polymorphism. Patients lacking activating KIR genes 3DS1 and 2DS1 were more common in severe RRP compared with mild-moderate RRP (78.8% vs 48.5%, p = 0.019). Further, patients carrying any of the known susceptible HLA-DRB1/DQB1 alleles were more frequently negative for KIR3DS1 (p = 0.006), KIR2DS1 (p = 0.003) or KIR2DS5 (p = 0.004) compared with controls carrying any of these HLA allotypes. Nearly 80% of the patients with severe disease were missing the protective HLA-DQB1*0602 allele as well as both KIR3DS1 and KIR2DS1 genes. Phenotyping of papilloma-infiltrating mononuclear-cells revealed an elevated numbers of NK cells and CD57(+)CD4(+) T cells in KIR3DS1(-)KIR2DS1(-) patients compared with patients carrying either one or both of these KIRs. Our data suggest that NK cells expressing activating receptors KIR3DS1 and KIR2DS1 may be necessary to trigger an effective early immune response against HPV-infected targets to establish resistance to RRP development.

Failure of gamma interferon but not interleukin-10 expression in response to human papillomavirus type 11 E6 protein in respiratory papillomatosis.

Recurrent respiratory papillomatosis (RRP) is a chronic, debilitating disease of the upper airway caused by human papillomavirus type 6 (HPV-6) or HPV-11. We describe responses of peripheral blood mononuclear cells (PBMC) and T cells from RRP patients and controls to the HPV-11 early proteins E6 and E7. PBMC were exposed in vitro to purified E6 or E7 proteins or transduced with fusion proteins containing the first 11 amino acids of the human immunodeficiency virus type 1 protein tat fused to E6 or E7 (tat-E6/tat-E7). T(H)1-like (interleukin-2 [IL-2], gamma interferon [IFN-gamma], IL-12, and IL-18), and T(H)2-like (IL-4 and IL-10) cytokine mRNAs were identified by reverse transcription-PCR, and IFN-gamma and IL-10 cytokine-producing cells were identified by enzyme-linked immunospot assay. These studies show that HPV-11 E6 skews IL-10-IFN-gamma expression by patients with RRP toward greater expression of IL-10 than of IFN-gamma. In addition, there is a general cytokine hyporesponsiveness to E6 that is more prominent for T(H)1-like cytokine expression by patients with severe disease. Patients showed persistent IL-10 cytokine expression by the nonadherent fraction of PBMC when challenged with E6 and tat-E6, and, in contrast to controls, both T cells and non-T cells from patients expressed IL-10. However, E7/tat-E7 cytokine responses in patients with RRP were similar to those of the controls. In contrast, E6 inhibited IL-2 and IL-18 mRNA expression that would further contribute to a cytokine microenvironment unfavorable to HPV-specific, T-cell responses that should control persistent HPV infection. In summary, E6 is the dominant inducer of cytokine expression in RRP, and it induces a skewed expression of IL-10 compared to the expression of IFN-gamma.