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Received: October 22^nd 2014

Accepted: May 15^th 2015

Escape mechanisms to the innate immune response in HPV-associated cervical cancer

Susana del Toro-Arreola,^a Mariel García-Chagollán,^a Luis Felipe Jave-Suárez^b

^aLaboratorio de Inmunología, Departamento de Fisiología, Centro Universitario de Ciencias de la Salud, Universidad de Guadalajara

^bDivisión de Inmunología, Centro de Investigación Biomédica de Occidente, Instituto Mexicano del Seguro Social

Guadalajara, Jalisco, México

Communication with: Susana del Toro-Arreola

Teléfono y fax: (33) 1058 5307

Email: susana@cucs.udg.mx

Cervical cancer is characterized by persistent human papilloma virus (HPV) infection. But, why, in some cases, is the immune system unable to reliably detect the HPV infection? For years, this has been a central question, which has yet to be fully answered. At present, it is well known that HPV has evolved a variety of mechanisms to evade the immune attack, and it is the success of these, which will be critical to determine whether the infection will be cleared or remain as a persistent infection. This review will be particularly focused on addressing some of the mechanisms used by HPV to avoid early recognition by the host innate immune system, which will then facilitate viral persistence with the consequent risk of eventual progression towards cervical cancer. Undoubtedly, an understanding of the balance between viral and immunological factors will provide crucial information that must to be taken into account for the design of prophylactic and therapeutic vaccines against HPV-associated cervical cancer.

Keywords: Immunity, Uterine cervical neoplasms, Papillomavirus infections.

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There are epidemiological and molecular data to support a causal relationship between infection with oncogenic types of human papillomavirus (HPV) and the development of cervical cancer (CC). However, HPV infection is, in most cases, a transient phenomenon resulting in the elimination of viruses without clinical evidence or leading to low-grade lesions that often show spontaneous regression. The presence of HPV is thus necessary but not sufficient for the development of cervical lesions; it is precisely the persistent infection that appears to be a prerequisite for the development of grade III cervical intraepithelial neoplasia and subsequently CC.^1-3 Environmental, viral, and host factors, as well as immune response, may influence the course of HPV infection;⁴ the latter factor plays an important role in determining if the infection will persist, turn back, or progress to more advanced lesions.^1,2,5 Hence the reason why the study of the mechanisms of immunity in CC has been largely directed towards HPV infection.

Although the immune response to HPV is not completely understood, it is known that local and systemic immune surveillance plays an important role during the establishment of latent infection. A latent infection can occur asymptomatically in approximately 30-60% of sexually active women. The interconnection between the mechanisms of innate immunity and antigen-specific immunity plays a critical role in defining elimination of the infection.^1-2,5 However, due to the infectious nature of the development of cervical cancer, it has been reported that there is a surprising number of women who do not develop an effective immune response to eradicate HPV infection.^1,6

The above findings indicate that HPV infection or the malignant transformation process somehow inhibit the ability of the immune system to generate an effective response.^5,7 Why does the immune system ignore or at least show failure to detect HPV infection? This has been a central question that has existed for years and is still unanswered in its entirety, even though today we know that HPV employs a variety of strategies to evade or subvert immune surveillance,⁷ which are critical to define whether viral persistence occurs or not, and therefore the risk of progression to cancer. Undoubtedly the understanding of the balance between viral and immunological factors will provide decisive information to be taken into account in the strategic planning of immunoprophylactic and therapeutic vaccines against cervical cancer associated with HPV infection.

Escape from natural barriers in cervical cancer associated with HPV infection

HPV replication is related to the differentiation of keratinocytes

Primarily, the HPV infectious cycle is already in and of itself a mechanism for immune evasion; this considers that it does not cause cell lysis, because the mechanisms of virus replication and release follow the differentiation program of the infected target cell itself, i.e. the keratinocyte,^8-10 which is already scheduled to die "of natural causes"; therefore, when a significant inflammatory process is not generated in the cervical epithelium, this death is not translated as a situation of harm, and it may result in persistent chronic infection. Certainly, signs of harm are a prerequisite for the migration of the first defense cells to the local environment, and that is how the innate system begins "the attack on the HPV-infected cell".^7-10

Langerhans cells undergo alterations in the HPV-infected epithelium

HPV infections being exclusively intraepithelial, viral attack should theoretically be initiated by antigen presenting cells (APC) in the squamous epithelium.^11,12 These sentinel cells (including epithelial cells, Langerhans, and dendritic cells) continuously "record" the microenvironment and with other innate effectors (monocytes, macrophages, polymorphonuclear leukocytes, and NK, or natural killer, cells) coordinate the protection of the mucosa of the cervical epithelium (Figure 1).⁷

In the cervical squamous epithelium, the Langerhans cells are the primary cell type responsible for antigen recognition, processing, and presentation,¹³ which makes these cells crucial in local immune defense because, once activated, they process the antigen and migrate to the nearest lymph nodes to present antigens to the CD4 + T lymphocytes; once this happens, the T lymphocytes already differentiated in effector cells are capable of activating in turn the CD8 + T lymphocytes, responsible for carrying out the destruction of infected keratinocytes.^7,13 However, HPV infection itself has been shown to decrease the number of cervical Langerhans cells, partly because the damage signals required to initiate the migration of antigen presenting cells are absent in the squamous epithelium associated with HPV infection, precisely because of the absence of cell death, as previously mentioned.⁸ It has also been seen that this decline is even more pronounced in cases that have progressed to intraepithelial neoplasia.^14,15

There is no evidence to support competent HPV replication in Langerhans cells, so a decrease in the number of these could be brought about indirectly through effects mediated by infected keratinocytes. At least in the case of HPV-16, it has been proposed that the E6 protein reduces the number of Langerhans cells in the epidermis by reducing E-cadherin on the surface of infected keratinocytes.¹⁴ E-cadherin plays an important role in the retention of Langerhans cells in the epidermis; it is thus through this pathway that HPV-16-infected keratinocytes limit the presentation of viral antigens by Langerhans cells, preventing the onset of an immune response mediated by T cells and, consequently, promoting viral persistency.^14,16,17

Another interesting focus in the immune microenvironment is the arrangement of molecules expressed on the surface of Langerhans cells, which constitutively express a battery of costimulatory molecules such as B7.¹⁸ Some studies have shown a very restricted costimulatory phenotype (CD11a, CD50, CD54, and CD86) in Langerhans cells present in samples of intraepithelial lesions, indicative of a limited immune response in the damaged cervical epithelium.^19-20 The phenotype of Langerhans cells in cervical neoplasia suggests, therefore, only limited immune activation in these lesions. In turn, it has also been reported that there are alterations in the expression of other important markers such as molecules of the major histocompatibility complex (MHC) class I (MHC-I) and class II (MHC-II),²¹ which are essential to carry out an efficient presentation of antigens to T lymphocytes.

MHC-II molecular disturbances favor the escape of cellular response

The ability to present exogenous antigens depends on the expression of MHC-II molecules. Typically, keratinocytes do not express MHC-II molecules, in contrast to how professional antigen presenting cells (such as Langerhans and dendritic cells) do. However, there are reports showing the transient expression of these molecules on the surface of keratinocytes derived from HPV-associated malignancies;²² as such, infected keratinocytes could gain the ability to present antigen to CD4 + T cells infiltrating the lesion.

Regarding the above, keratinocytes transfected with the E5 protein of HPV-16 have been shown to have a reduced acidification in endocytic compartments (where proteolytic degradation of exogenous antigens is performed), which results in poor generation of antigenic epitopes, with consequent alteration in the formation of mature MHC-II complexes.²³ Additionally, poor acidification in endosomes also prevents degradation of the invariant chain (Ii), a major chaperone in the maturation of MHC-II complexes, digested by proteases that work only in acid pH, which leads to inhibition of the expression of MHC-II molecules on the cell surface (Figure 2), which may allow the infected cells to escape immune recognition of the host and consequently promote establishment and persistence of HPV.²⁴

Defects in MHC-I molecules in cervical epithelium

In another vein, a loss in the expression of MHC-I molecules has also been reported in CC.²⁵ In different studies of cervical specimens from patients with cervical squamous cell carcinoma, partial or complete loss of expression of MHC-I molecules has been observed,²¹ where reduced expression was seen in up to 70% of cases (with loss of a monomorphic region or even loss of a specific allele).¹⁷ The absence of MHC-I molecules in CC has been explained through various mechanisms, including changes in processing machinery and assembly of mature MHC-I complexes. For example, one molecule involved in the MHC-I presentation pathway is the TAP protein (transporter associated with antigen processing), composed of heterodimer TAP1/TAP2. Its function is to deliver peptides derived from cytosolic antigen to the lumen of the endoplasmic reticulum, where these peptides will be associated with MHC-I molecules. Thus, any defect in the TAP protein could significantly alter antigen presentation. Precisely the defects in genes encoding the heterodimer TAP1/TAP2 are common in CC, in which it has been seen that this failure correlates with disease progression.²⁶ In addition to the genetic alterations described, it has also been seen that the E7 oncoprotein of HPV-18 represses the promoter activity of TAP1, which is reflected in transcriptional inhibition of TAP1.²⁷ These findings indicate that CC is under selective pressure and they suggest keeping cells deficient in MHC-I molecules. Thus, the deregulation of expression of these molecules on the surface of keratinocytes facilitates HPV persistence and lesion progression to more advanced stages, precisely by promoting the escape from cytotoxic attack mediated by CD8 + T cells (which are the primary cell type in the defense against viral infections and tumor growth control). 

The above information indicates that CC is under a degree of immune surveillance in which at least the immune response mediated by cytotoxic T lymphocytes would not then be the most effective for cases in which the tumor cells have stopped expressing class I histocompatibility molecules. Thus, according to the missing self-hypothesis proposed by Kärre in 1990, a function of NK cells is to recognize and eliminate cells that have ceased to express MHC-I molecules (an event frequently observed in tumor cells or cells infected by viruses).²⁸ Consequently, all of the above findings could be clinically relevant, considering that during CC development, expression of MHC-I molecules is lost, so theoretically these cells would be a suitable target to be eliminated by NK cells. However, alterations in NK cells have also been described in patients with cervical cancer.  

CC is not a suitable target for lysis by NK cells

The role of NK cells in the development of cervical cancer is not fully elucidated, although the last two decades have seen great progress in understanding the mechanisms that regulate their activity, which is under a fine control of signals generated by activation and inhibition receptors that, collectively, dictate the destination of NK cells in natural cytotoxicity against virus-infected cells or transformed cells.²⁹

An important group of receptors with the ability to inhibit NK cell-mediated cytolysis are killer immunoglobulin-like receptors (KIR). The biological significance of KIR is precisely preventing the death of normal cells expressing MHC-I molecules, but once these have ceased to be expressed, they become a suitable target for elimination by NK cells.²⁹ The importance of the KIR repertory has not yet been explored in CC, and the few findings about it are reported in a study in which the presence of certain KIR genotypes was evaluated, and it was found that certain genes and genotypes are associated with an increased risk of developing cervical neoplasia.³⁰ However, the biological significance that these findings may have specifically for HPV infection, and their association with the development of cervical cancer, has not been fully defined. 

Furthermore, NK cells can be efficiently activated after recognizing tumor or virus-infected cells that had previously been covered with antibodies. This recognition by CD16 (low-affinity receptor for the Fc portion of IgG1 and IgG3) results in the destruction of the infected cell through a mechanism known as antibody-dependent cellular cytotoxicity (ADCC). Thus, it is clear that ADCC is a potentially important pathway for immune recognition of tumors and virus-infected targets. However, an inability of NK cells derived from patients with CC to eliminate tumor cells has also been demonstrated. Interestingly, these studies also demonstrated that the decrease in natural cytotoxicity was more severe as tumor burden and disease stage grew.³¹

Besides ADCC, NK cell function undoubtedly also depends heavily on a new family of receptors called NCR (natural cytotoxicity receptors). This family includes three members: NKp30, NKp46, and NKp44; the first two is constitutively expressed in NK cells; the latter only in activated cells. Interestingly, it has been found that NK cells from cancer patients show decreased phenotype expression of NCR. This has been observed particularly in patients with acute myeloid leukemia, from which it has been shown that alterations in the natural cytotoxic function occurred due to poor expression of NCR.³² The significance of NCR during the development and progression of CC has not been explored in depth; however, a decrease in expression of NKp30 and NKp46 has been reported in peripheral blood NK cells of women with CC and high-grade lesions; in addition, this defect is associated with low cytolytic activity of these cells.³³ Another interesting finding in this regard has to do with the NK cells of patients with precancerous lesions or cancer of the cervix. These cells show defects in the expression of components involved in signaling pathways. Such has been the case of defects observed in the expression of the zeta chain,³⁴ which may have biological implications, because at least in the case of NKp30 and NKp46, these receptors are associated with this chain in order to transmit the signal generated from the extracellular medium into the cell, in a similar mechanism to that of the T cell receptor (TCR) in T lymphocytes.³⁵ Thus, defects in cell-signaling suggest that even at early stages of cervical lesions, some extension of immune suppression reflected by the decrease in the zeta chain expression has already occurred, which could be caused by neoplastic lesion or by the HPV infection per se.   

Conclusions

So far, it is clear that although some persistent infections with oncogenic HPV types may progress to CC, the host’s immune response will generally be able to eliminate the majority, which provides an opportunity to prevent this tumor through vaccination programs. This review addressed some of the mechanisms used by HPV to subvert immune recognition and, certainly, this virus must have additional mechanisms that, in short, allow it to be permanently ignored by the immune system. To the extent that new mechanisms are discovered by which cervical lesions associated with HPV infection escape local immunosurveillance and progress to cancer, it will be possible to have more effective means for tumor prevention. For this reason, cervical cancer control programs need to be reassessed to take into consideration factors such as a satisfactory tolerance to the vaccine, and making sure that it provides adequate immunogenicity to reverse the strategies used by HPV to avoid the immune response. Once these barriers are addressed, cervical cancer vaccination will be able to greatly reduce medical costs generated, but more importantly, will reduce the number of deaths due to this tumor.

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