Cervical cancer affects approximately 500,000 women each year, and almost all cases are associated with HPV infection (4
). The immune response elicited by HPV includes the production of specific antibodies as well as a cell-mediated response. However, persistent HPV infection occurs in 10 to 20% of infected individuals due to a series of immune evasion mechanisms (24
). Although HPV infects basal keratinocytes, high-level viral protein expression and viral assembly are usually limited to the upper layers of epithelia, thus avoiding contact with cells of the immune system, such as Langerhans cells. In addition, assembly of HPV virions or self-assembly of L1 into VLPs is required for efficient production of L1-specific IgG1 and IgG2 antibodies (25
). Thus, HPV-specific antibodies are not detected in a large proportion of infected individuals. Nevertheless, clearance of HPV is accompanied by antibody production directed against L1 antigen, and passive transfer of specific antibodies in animal models has been shown to confer immunity against infection (25
). Likewise, the protection against HPV induced by the L1 vaccine is associated with production of antibodies specific for this antigen (24
). On the other hand, cell-mediated immunity also has a relevant role in the control of HPV infection. Thus, the development of an immune response mediated by CD4+
Th1 lymphocytes has been associated with regression of ano-genital warts (21
). In addition, the increased frequency of cervical cancer in patients with altered CD4+
cells highlights the importance of this cell population in the control of HPV infection (23
Evasion of the innate immune system is thought to play a key role in the ability of HPV to cause persistent infection. This is reflected in the absence of an inflammatory response to the virus, as well as downregulation of interferon release (12
). In addition, several studies have suggested that evasion of NK cell activity may play a role in the development of cervical cancer (3
). In this regard, Carrington et al. found that the presence of the KIR3DS1 gene is a risk factor for developing cervical cancer (3
). In addition, a reduction in the expression of NKp30, NKp46, and NKG2D by NK cells of women with cervical cancer has been reported (2
). It has also been shown that HPV E6 and E7 proteins interfere with the binding of IL-18 to its receptor, resulting in a reduction in IFN-γ production by NK cells (12
). Furthermore, in different malignant tumors the expression of HLA-G, which interacts with the inhibitory receptors ILT2, ILT4, and KIR2DL4, has been detected, and this has been suggested as a mechanism leading to evasion of NK cell activity (22
). In this regard, increased expression of HLA-G in patients with cervical malignancies has been reported (30
) as well as reduced synthesis of some activating ligands for NK cell receptors (26
). Altogether, these studies suggest that modulation of the expression of NK receptors and their ligands affects the immune response to HPV. However, the precise role of different NK cell receptors in this context has not been fully defined. Furthermore, there is little information regarding the response of NK cells to HPV vaccination.
In this study, we have detected increased expression of NKG2A, NKG2D, NKp30, and NKp46 by NK cells and of ILT2 by monocytes, NK, and CD3+
lymphocytes after HPV vaccination. In addition, an increase in the density of expression (MFI) of ILT2, NKG2D, NKp30, and NKp36 was also observed. ILT2 is a negative regulatory receptor that has been shown to participate in the immunopathogenesis of several autoimmune diseases (5
). Furthermore, an increase in the expression of this receptor by NK and T lymphocytes has been shown in association with HCMV infection (8
). Although the precise role of ILT2 under this condition has not been fully elucidated, it is very feasible that increased expression of this receptor may contribute to downregulating the immune response against HCMV. In this regard, it has been reported that cytotoxic T cells may express ILT2 and that this receptor modulates their lytic activity (10
). Thus, our data showing an increase in the expression and function of ILT2 after quadrivalent HPV (type 6/11/16/18) vaccine immunization strongly suggest that this receptor may also have a relevant role in the modulation of the innate and adaptive immune responses against HPV. Interestingly, we have observed that the enhanced synthesis of IFN-γ observed in vitro
upon quadrivalent HPV (type 6/11/16/18) vaccine immunization is not affected by ILT2 engagement, indicating that the regulatory effect of this receptor is not exerted on all immune parameters that are relevant in the response to HPV. In this regard, it has been recently reported that NK cells exposed to HPV VLPs show increased cytotoxic activity and cytokine production (including IFN-γ) (19
). Therefore, it is possible that the enhanced production of IFN-γ observed by us upon HPV immunization could be related to the increased expression of activating receptors by NK cells and to a lack of a regulatory effect mediated by ILT2 on this immune parameter. In this regard, the possible effect of quadrivalent HPV (type 6/11/16/18) vaccine immunization on the cytotoxic activity of NK lymphocytes remains an interesting point to be addressed.
Interestingly, we have observed that while the proportion of CD8+ lymphocytes that expressed ILT2 decreased after immunization, there was an increase in the density (MFI) of this receptor in these cells. Although the underlying mechanism for this apparent paradoxical effect remains to be determined, our data suggest that in the fraction of CD8+ cells that remain ILT2 positive after quadrivalent HPV (type 6/11/16/18) vaccine immunization, the enhanced density of this receptor may exert an increased regulatory effect.
The in vitro
effect of the quadrivalent HPV (type 6/11/16/18) vaccine on the expression of ILT2 by NK cells after HPV immunization is of interest. It is feasible that the induction of ILT2 expression by quadrivalent HPV (type 6/11/16/18) vaccine in these cells observed by us corresponds to the memory-like phenomenon described under different circumstances for NK lymphocytes (17
). However, it is also possible that this induction is mediated by cytokines released by T cells. Finally, the adjuvant contained in the vaccine (aluminum) might also contribute to the induction of ILT2.
Overall, the profile of NK receptors displayed by NK cells after immunization differs significantly from changes observed in association with persistent HPV infection and cervical cancer (7
). This may reflect differences in the route of antigen exposure, an effect produced by the combination of antigen and adjuvant contained in the vaccine, the absence of the effects produced by HPV-E6 and −E7, or genetic host-related factors.
In summary, our observations indicate that HPV immunization is associated with significant changes in the expression and function of immune innate and regulatory receptors, phenomena that may contribute to the protective effect of this vaccine.