Human genetic and immunological dissection of papillomavirus-driven diseases: New insights into their pathogenesis

Abstract

Human papillomaviruses (HPVs) are responsible for cutaneous and mucosal lesions. Persistent HPV infection remains a leading cause of uterine cancer in women, but also of cutaneous squamous cell carcinoma in patients with epidermodysplasia verruciformis (EV), and of rare and devastating benign tumors, such as “tree-man” syndrome. HPV infections are usually asymptomatic or benign in the general population. Severe manifestations in otherwise healthy subjects attest to inherited immunodeficiencies. The human genetic dissection of these cases has identified critical components of the immune response to HPVs, including the non-redundant roles of keratinocyte-intrinsic immunity in controlling β-HPVs, and of T cell-dependent adaptive immunity for controlling all HPV types. A key role of the CD28 T-cell costimulation pathway in controlling common warts due to HPVs was recently discovered. This review summarizes the state of the art in the human genetics of HPV infection, focusing on two key affected cell types: keratinocytes and T cells.

Introduction

Human papillomaviruses (HPVs) are non-enveloped viruses with ~8 kb double-stranded circular DNA genomes packaged into an icosahedral capsid. They display strict tropism for skin and squamous stratified epithelia. They first infect basal stem cells and complete their cycle during the course of keratinocyte differentiation in successive layers. HPVs have been classified into five genera, including over 200 HPV genotypes, according to the International Human Papillomavirus Reference Center (Papillomavirus Episteme (PaVE); https://pave.niaid.nih.gov/#home) [1]. HPVs of the β, γ, μ and ν genera display strict cutaneous tropism, whereas α-HPVs include both cutaneous and mucosal specific types. β-HPVs generally cause asymptomatic infections, but are responsible for pytiriasis versicolor-like lesions and flat warts, and non-melanoma skin cancer (NMSC) in rare patients with epidermodysplasia verruciformis (EV) [2]. γ-, μ- and ν-HPVs cause benign cutaneous common and plantar warts. Different types of α-HPVs are associated with cutaneous warts, benign mucosal lesions and carcinomas of the anogenital and oropharyngeal tracts [3,4]. The vast majority of individuals within the human population have been exposed to HPVs, as demonstrated by serological data [5–8]. Infection is mostly asymptomatic or benign and self-healing [9]. The onset of HPV-induced cancer can be attributed primarily to persistent lesions, due to a failure to clear replicative infection. For instance, once infected, less than 10–20% of women fail to clear cervical lesions spontaneously within two years, revealing strong interindividual variability in host immunity to HPVs [4,10].

Severe HPV infections of all types are frequent in patients with acquired immunodeficiencies, whether due to human immunodeficiency virus (HIV) infection or immunosuppressive treatment, suggesting an important role for adaptive T-cell immunity, and CD4⁺ T cells in particular, in the control of HPV infection [11–14]. However, many patients without acquired immunodeficiencies suffer from severe HPV infection, suggesting a congenital immune defect. The characterization of inborn errors of immunity (IEI) in these patients provides an interesting approach for the study of non-redundant immune mechanisms for the effective control of HPV infection. These studies have already revealed an important role for tissue-specific and cell-intrinsic immunity in keratinocytes, and key molecular mechanisms for protective T-cell immunity against HPVs. We review here the knowledge acquired from the study of these IEI, with particular focus on the two key cell types affected: keratinocytes and T cells. Indeed, the underlying defects range from inborn errors of keratinocyte-dependent intrinsic immunity to inborn errors of T cell-dependent adaptive immunity affecting both T cells and antigen-presenting cells (APCs). Neither known disorders of B-cell immunity nor defects of myeloid or lymphoid leukocyte-mediated innate immunity are associated with a predisposition to HPV lesions.

1-. Keratinocyte intrinsic immunity

Viruses generally display cell-specific tropism, and HPVs display a particular tropism for keratinocytes. The ability of target cells to restrict viral replication and assembly directly and/or to signal their “infected” status to immune cells, both of which define cell-intrinsic immunity, is crucial for infection control. Cell-intrinsic immunity can operate in any of the > 400 discernable hematopoietic and non-hematopoietic cell types of the human body. The development and persistence of HPV lesions reflect a two-step mechanism in which keratinocytes are permissive to replicative HPV infections, and T cells fail to clear infected keratinocytes. β-HPVs are frequently found in the skin of healthy individuals, but copy numbers are generally low, and they do not cause visible lesions. By contrast, EV patients have a high viral load and visible lesions [15–17]. Three IEI have been identified in patients with isolated EV: EVER1, EVER2 and CIB1 deficiencies [2,18,19]. EVER1, EVER2 and CIB1 form a complex that restricts β-HPV infection in keratinocytes. This keratinocyte-intrinsic viral restriction accounts for the lack of symptomatic β-HPV infection in healthy individuals, and the occurrence of such infections in EV patients. ZnT1, a zinc transporter, interacts with both EVER proteins, but not with CIB1. Consistent with this finding, abnormal zinc homeostasis is observed only in EVER-deficient cells, suggesting that EVER proteins have functions other than HPV restriction that are CIB1-independent and not involved in EV pathogenesis. Furthermore, CIB1-deficient keratinocytes have no particular abnormal migration, cell growth or adhesion phenotype. The EVER/CIB1 complex is targeted by the viral E5 and E8 proteins expressed by α-HPVs and γ-, μ- and ν-HPVs, respectively, but not by β-HPVs, suggesting that E5 and E8 function, at least partly, to overcome EVER/CIB1-dependent restriction. The precise molecular mechanism by which β-HPV replication is controlled by the EVER/CIB1 complex in keratinocytes remains to be established, but we hypothesize that the lack of a functional EVER/CIB1 complex compensates for the missing E5/E8 proteins, suggesting a role for this complex in a regulatory pathway controlling the division of infected epidermal stem cells.

2-. Defects of APCs and T cells

a. Immune infiltrates in regressing warts

Human and animal studies of regressing or inflamed warts have provided clues to the cells involved in the antiviral effector phase [20–27]. In dogs infected with COPV (canine oral papillomavirus), the lesions start to regress eight weeks post-infection, and this regression is associated with an infiltration of CD4⁺ and CD8⁺ T cells, and dendritic cells (DCs) into the infected epithelium and the underlying lamina propria [24]. In rabbits, the regression of skin lesions caused by CRPV (cottontail rabbit virus) is mediated by CD8⁺ T cells, but not CD4⁺ T cells [23]. The most detailed study of HPV-induced lesions in remission to date concerned HPV6⁺ and HPV11⁺ anogenital warts and was published in 1994. This study showed an infiltrate of leukocytes, predominantly CD4⁺ and, to a lesser extent, CD8⁺ cells, in the epidermis and dermis of the regressing warts, and an accumulation of macrophages in the dermis under the lesion [22]. The findings of this study were consistent with those of a 1986 study of planar wart regression, showing a dermal and epidermal infiltrate constituted principally of CD4⁺ cells [20]. Recent studies of regressing cutaneous warts have reported a major dermal infiltrate of poorly characterized DCs, including a few plasmacytoid DCs (pDCs), and a lymphocytic infiltrate in the dermis below the lesion, consisting mostly of CD8⁺ T cells, contrasting with the results of previous studies [25,27]. One possible explanation for this discordance may be the expression of CD4 on some myeloid cells undistinguishable from CD4⁺ T cells by immunohistochemistry. Together, these studies suggest a major role for T cell-mediated immunity in the control of HPV infection.

b. Hypomorphic mutations of severe combined immunodeficiency genes

Given the importance of immunological infiltrates in wart regression, it was no surprise to find that IEI affecting adaptive cell responses were associated with severe HPV infections, provided of course that they were compatible with survival beyond infancy. Indeed, although PCR-based studies detect β- and γ-HPV DNA on skin swabs as soon as the first days/weeks of life [16], it should be borne in mind that HPV-driven lesions are rare in young children. The prevalence of cutaneous warts gradually increases in children of school age, peaking at 10–15 years of age [28]. The prevalence of anogenital warts is highest between 20 and 30 years of age [29]. Diseases such as reticular dysgenesis or severe combined immunodeficiency (SCID), in which T-cell development is abolished, have not, therefore, been found to be associated with HPV infection, because the patients die before the age of one year in the absence of hematopoietic stem cell transplantation (HSCT) [30]. By contrast, hypomorphic mutations of SCID genes allowing some T-cell development and survival into adulthood have been associated with a high degree of susceptibility to HPVs and many other pathogens. The mutations concerned include autosomal recessive (AR) hypomorphic mutations of the ADA [31,32], CORO1A [33–35], DCLRE1C [36,37], JAK3 [38], LIG4 [39,40], RAG1 and RAG2 [41,42], and ZAP70 [43] genes, and X-linked recessive (XLR) mutations of IL2RG [44–46].

c. Other IEI impairing T-cell development or migration

Other IEI partially impairing T-cell development have also been associated with severe cutaneous warts. These IEI include biallelic mutations of the TRAC [47], NHEJ1 [48], ATM [49], LCK [50], IL7 [51,52] and STK4 [53,54] genes, and hemizygous mutations of the SASH3 gene [55]. Deficiencies of TRAC, NHEJ1 and ATM partially impair T-cell receptor (TCR) and/or B-cell receptor (BCR) rearrangement. Deficiency of SASH3 impairs thymocyte survival. LCK is a signal-transducing molecule acting downstream from several T-cell costimulation molecules, such as CD4, CD8 and CD28. IL-7 deficiency partially impairs T-cell development, whereas complete loss-of-function (LOF) mutations of the genes encoding its two coreceptors, IL7R and IL2RG, are associated with a complete absence of T cells. STK4 is important for T-cell development, and studies in mice have shown that peripheral naïve T lymphopenia is due to impaired thymic egress [56,57]. Deficiencies of LCK, IL-7, SASH3 and STK4 lead predominantly to CD4⁺ T-cell lymphopenia. Two inherited immunodeficiencies with a very high penetrance for severe HPV infection are caused by autosomal dominant (AD) gain-of-function (GOF) CXCR4 [58–60] and AD LOF GATA2 [61–65] mutations. These immunodeficiencies are associated with severe cutaneous or anogenital wart in 80% and 50% of patients, respectively. Unlike the other IEI mentioned above, they simultaneously compromise multiple leukocyte subsets, notably reducing APC and T-cell counts, particularly for CD4⁺ T cells. The important role of myeloid cells in the susceptibility to HPV of CXCR4-deficient patients was confirmed by the remission of warts in a patient who spontaneously lost the CXCR4 GOF allele in the myeloid compartment [66]. Consistent with the importance of both APCs and T cells, AR DOCK8 deficiency is associated with a combined immunodeficiency characterized by severe viral infections of the skin and mucosae, including warts in >40% of the patients [67–69]. In particular, DOCK8 plays a crucial role in optimal dendritic cell migration to lymph nodes, and in the survival of T cells in collagen-dense tissues [67,70,71]. Its deficiency therefore impairs both priming and effector anti-HPV immune responses. In addition to its role in thymic egression, STK4 promotes T-cell migration via DOCK8 recruitment [57], possibly contributing to skin and mucosal viral infections in STK4 deficient patients. Thus, studies of IEI show that low counts of APCs and T cells, and the impairment of APC and T-cell migration confer a predisposition to severe HPV infection.

d. IEI impairing optimal TCR activation or costimulation

Other IEI underlying HPV lesions have only a modest impact on the counts of T lymphocytes and APCs. These disorders inform us about specific molecular events required for HPV clearance. Mutations impairing signaling downstream from the TCR also confer a predisposition to HPV infection. AR RHOH deficiency is associated with multiple conditions, including EV. RHOH regulates LCK/ZAP70 signaling in the TCR pathway [72]. A biallelic missense mutation of TAOK2 has been reported in two patients with severe warts and normal T-cell counts but impaired TCR activation [73]. TAOK2 activates the MAP kinase pathway and regulates cytoskeleton dynamics, but its role in T cells remains unknown [74]. More recent findings have suggested that some other T-cell coreceptors are more important for the control of HPV infection. Biallelic LOF mutations of CD4 have been reported in a patient with isolated severe warts [75]. CD4 is a coreceptor for human leukocyte antigen II (HLA-II). It increases positive selection in the thymus and the antigen response of CD4⁺ T cells [76–78]. However, CD4 is also expressed on myeloid cells, and could therefore also play a role in APCs. We recently reported CD28 deficiency in three patients with severe isolated verrucosis, including one patient with “tree man” syndrome [79]. CD28 is a major costimulation molecule in TCR signaling, recognizing CD80 and CD86 on APCs. Our findings suggest that the CD28 costimulation pathway plays an important role in controlling the HPVs responsible for cutaneous common warts. About 30% of patients with AR CARMIL2, AD CARD11 or XLR MAGT1 deficiency have severe cutaneous or anogenital warts, in addition to several other infections [80–85]. In light of the discovery of patients with CD28 deficiency, the susceptibility to HPV of these patients can now be explained by a CD28 signaling defect. CARMIL2 deficiency specifically impairs CD28 signaling in mouse and human T lymphocytes [80,86]. MAGT1 is involved in magnesium regulation and protein glycosylation. Its deficiency results in a glycosylation defect that, among its over effects, impairs CD28 expression on T lymphocytes. Dominant-negative CARD11 mutations impair NF-κB activation upon CD3/CD28 costimulation, and CARD11 is a direct partner of CARMIL2 [87,88]. Together, given the absence of warts in patients with IEI affecting other T-cell costimulation receptors (e.g. CD27, ICOS, CD40L), these recent discoveries suggest a non-redundant role for CD4 and CD28 costimulation in the control of the virus.

Conclusion

IEI have provided important clues to improve our understanding of the immune response to HPVs. First, despite a clear preventive effect of anti-HPV vaccination, inborn errors of adaptive B-cell immunity do not confer a predisposition to severe HPV infection. Likewise, IEI of leukocyte (myeloid or lymphoid) innate immunity (affecting the oxidative burst, for example) are not associated with overt HPV infections. It is now clear that the major contributing branches of immunity are keratinocyte-intrinsic immunity for β-HPVs, and T cell-dependent adaptive immunity for all HPV types. CD4⁺ T cells clearly play a central role, as highlighted by severe HPV infections in patients with HIV-mediated acquired immunodeficiency or IEI resulting in selective CD4⁺ T-cell lymphopenia, and the recent discovery of CD4 deficiency in a patient with generalized verrucosis. IEI affecting the Th1 (IL-12/IFN-γ) and Th17 (IL-17) axes of CD4⁺ T-cell immunity are not associated with HPV disease, and have instead been shown to be associated with susceptibility to mycobacterial and fungal infections, respectively [89,90]. Future studies should decipher the functional component of the CD4⁺ T-cell response required for the control of papillomaviruses. A key role for the CD28 costimulation pathway has recently emerged, with the implication of CD28, CARD11, CARMIL2 and MAGT1 deficiencies, all of which impair CD28 expression or signaling in T cells. Finally, patients with SCID due to IL2RG or JAK3 deficiencies may develop EV, or other recurrent and persistent warts, despite successful HSCT and full correction of their T-cell immune defect [91–93]. Conversely, patients with SCID due to other IEI (e.g. RAG1 or RAG2 deficiencies) are not prone to the development of HPV infections after HSCT. It is tempting to speculate that the post-transplantation susceptibility to HPV of patients with IL2RG and JAK3 deficiencies is skin-intrinsic, affecting either keratinocytes or Langerhans cells, the only professional APCs in the epidermis, or both [94]. HSCT in patients with SCID will constitute an interesting model in which to extend our knowledge of the skin-intrinsic mechanisms at work in the control of HPV infection.

Figure.

Inborn errors of immunity (IEI) conferring predisposition to severe HPV infections impair either keratinocyte-intrinsic immunity or adaptive T-cell immunity at the priming or effector level. APC: antigen presenting cell.