Loss of CD28 within CD4+ T cell subsets from cervical cancer patients is accompanied by the acquisition of intracellular perforin, and is further enhanced by NKG2D expression
Introduction
Cervical cancer (CC), which typically develops from pre-existing non-invasive squamous precursor lesions [1], still remains as one of the most common causes of cancer death among women in the developing world [2], [3]. Long-term persistent infection with high-risk human papilloma virus (HPV) strains is recognized as the strongest causative agent of CC [4], [5], [6]. Although the incidence of genital HPV infection is very frequent in young sexually active women [7], the majority of the cervical lesions are self-limited and spontaneously regress within 12–24 months post-diagnosis of the dysplastic smear [8], [9], [10]; only in a very low percentage of women will HPV infection evolve into low and/or high-grade lesions and progress eventually into cancer [11], [12], [13]. HPV-associated lesions are usually cleared by the result of an effective T cell-mediated immune response directed against early viral proteins [14], [15] with CD4+ and CD8+ T cells more likely to be the major effector cells involved in HPV clearance [16], [17]. This idea has been well exemplified by studies that have shown that spontaneously regressing genital warts are histologically accompanied by a CD4+ T cell response [18], [19], [20]. Therefore, there is no doubt that CD4+ T cells are essential regulators for the control of HPV-related cervical lesions, preventing their progression towards cancer.
For optimal effectiveness and specificity, the mammalian immune system has evolved to have naïve CD4+ T cells under the control of different triggering signals. Basically, T cell activation can be visualized as occurring in two phases, the first phase lies in the specific recognition of the antigenic peptide presented via MHC molecules on the surface of professional antigen presenting cells, triggering the master T cell receptor (TCR); the second phase is provided by co-stimulatory signals which complement the TCR signaling pathway [21], [22]. CD28 on naïve T cells is by far the most effective co-stimulatory receptor and it is considered an essential co-stimulatory-signaling molecule that determines the quality and quantity of T cell immune responses [23], [24].
Nearly all CD4+ T cells of healthy, normal individuals exhibit cell surface expression of CD28, which is critical for T cell activation, proliferation and survival processes. TCR activation in the absence of a co-stimulatory CD28 signal may lead T cells into a state unresponsive to subsequent re-stimulation [22], [25], [26]. However, CD4+ T cell responses may not necessarily be dependent on CD28-mediated co-stimulation. This is the case in some chronic inflammatory scenarios, wherein there has been observed an expansion of CD4+CD28null T cells. This atypical population has been a theme of extensive immunological research; for instance, one of the first experimental publications that substantiated the existence of CD4+ T cells with a lack of CD28 arose from studies in patients with rheumatoid arthritis, where an expansion of the CD4+CD28null T cell compartment was observed, which was at least partially due to the emergence of oligoclonal T cell populations [27]. Similarly, Markovic-Plese et al. characterized a subpopulation of CD4+ T cells with a long-term stable CD28null surface phenotype in patients with multiple sclerosis. Importantly, this T cell population exhibited an increased survival after activation, which was likely related to the lack of surface CTLA-4 [28]. Since these publications, numerous studies have revealed the occurrence of a significant number of CD4+CD28null T cells in other inflammatory circumstances, such as those caused by chronic viral infections.
The biological function of this particular population has not been wholly unravelled at present; however, the absence of the CD28 co-stimulatory molecule has been associated with a significant increase of intracellular perforin and granzyme B [29]. This observation suggests that the CD4+CD28null T cell compartment may have a cytotoxic capability, reminiscent to that used by NK cells to lyse target cells; this suggests that CD28null T cells may remain functionally active even when they lack CD28-mediated co-stimulation. Interestingly, it has been observed that CD4+CD28null T cells not only acquire cytotoxic proteins, but also acquire the expression of other receptors commonly expressed by NK cells, for example, the activating receptor NKG2D [30], [31]. NKG2D, which serves as a major receptor for the sensing of self-molecules induced by cellular stress, was originally described as being restricted to NK cells and CD8+ T cells; that is, lymphocytes with major lytic activity. However, the discovery of an unusual subset comprised of CD4+ T cells that do express NKG2D in patients with autoimmune diseases, and that apparently promote damage [32] challenged the original idea that this receptor was strictly confined to cytotoxic cells.
Nevertheless, paradoxical data have shown that this unusual population may also be armed with immunosuppressive properties in cancer patients [33] and the lack of CD28 has also been tagged with a role in the age-associated immune function decline [34]. This confusion has led to the proposal that two distinct populations of CD4+NKG2D+ T cells might exist: one with a classical cytotoxic role, the other one with an immunoregulatory role. Interestingly, in one of our previous studies, we showed a significant increase of CD4+NKG2D+ T cells in patients with cervical intraepithelial lesions, including cancer [35], [36], and we speculated that this increase might be the result of chronic exposure to viral and/or pro-inflammatory factors. More recently, we have revealed the existence of two separate CD4+NKG2D+ T cell populations, which were defined by the presence or absence of the co-stimulatory molecule CD28, with the CD4+NKG2D+CD28null population more frequently observed in women with established cervical cancer. This has led to the present effort to further characterize this population in patients with cervical cancer; we wonder if it is the loss of CD28 that indeed influences the acquisition of cytotoxic properties or if, conversely, it is the presence of NKG2D which might influence the CD4+ T lymphocytes to adopt a regulatory role. Here, we provide the first evidence of a strict link between the absence of CD28 and the expression of perforin, which is likewise enhanced by the expression of NKG2D, within selected CD4+ T cells from cervical cancer patients.
Section snippets
Patients and healthy donors
We studied ten samples from women with a colposcopic and histopathologic diagnosis of invasive squamous cervical carcinoma (CC), median age 53 years (range 36–77). In order to study our population of interest, and based on our previous results [36], only samples with more than to 4% of CD4+NKG2D+ T cells from total CD4+ T cell population by flow cytometry were chosen for inclusion in this study. As a control group, we included ten age/gender-matched samples from healthy women with normal Pap
Expansion of CD4+CD28null T cells, many of them NKG2D+, was strikingly observed in selected cervical cancer (CC) samples compared to controls
As described in our previous report [36], CC patients frequently showed an expansion of the circulating CD4+NKG2D+ T cell population. In accordance with this increase, the majority of CD4+NKG2D+ T cells did not express CD28. In this current work, we examined the frequency of CD4+ T cell subsets defined by the presence or absence of CD28 and NKG2D in ten control and ten CC samples, which were selected based on CD4+NKG2D+ T cell percentages (Fig. 1). We found an expansion of CD4+CD28null T cells
Discussion
It has been more than 30 years since the acquisition of lytic functions within the repertoire of CD4+ T cells was first reported [37]; however, these first observations were largely restricted to clones generated from in vitro CD4+ T cell cultures. Therefore, skepticism arose and it was argued that such CD4+ T cells with cytotoxic capacity were more likely to represent an in vitro artifact, without truly having a physiological role. More recently, some studies have suggested that the loss of
Conclusions
Our data provide the first evidence of a strict link between the absence of CD28 and the expression of perforin, which is likewise enhanced by the expression of NKG2D, within selected CD4+ T cells from cervical cancer patients. However, the mechanism, which controls or links the absence of CD28 and the presence of NKG2D with perforin expression, should be investigated in the near future. The expanded cervical cancer CD4+CD28null T cell subsets lacked any of the classical immunoregulatory
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
MES performed all the experimental work, searched scientific literature, contributed with figures and wrote the manuscript. MRBT and ALPS assisted with scientific thinking and experimental work. EGB and JH helped with data analyses and assisted with proofreading of the manuscript. LFJS and AAL contributed with multiparameter flow cytometry assistance and collection of cancer samples. JGF and NVM contributed with experimental work. STA conceived and designed the theoretical framework of the
Acknowledgements
This work was supported by grants from the Consejo Nacional de Ciencia y Tecnología through Convocatoria para el Fondo Sectorial de Investigación en Salud y Seguridad Social SS-IMSS-ISSSTE-CONACYT (2012-182791) and Convocatoria de Proyectos de Desarrollo Científico para atender Problemas Nacionales (2015-993), both to STA.
References (55)
- et al.
Local immune response in the microenvironment of CIN2–3 with and without spontaneous regression
Mod. Pathol.
(2010) - et al.
Immunohistochemical analysis of CD4+ and CD8+ T-cell subsets in high risk human papillomavirus-associated pre-malignant and malignant lesions of the uterine cervix
Gynecol. Oncol.
(2006) The expanding world of co-stimulation: the two-signal model revisited
Trends Immunol.
(2001)- et al.
Role of the CD28 receptor in T-cell activation
Immunol. Today
(1990) - et al.
The CD28 family: a T-cell rheostat for therapeutic control of T-cell activation
Blood
(2005) - et al.
CD28(−) T cells: their role in the age-associated decline of immune function
Trends Immunol.
(2009) - et al.
Pathogenic features of CD4+CD28- T cells in immune disorders
Trends Mol. Med.
(2012) - et al.
Preferential utilization of the perforin/granzyme pathway for lysis of Epstein-Barr virus-transformed lymphoblastoid cells by virus-specific CD4+ T cells
Virology
(2001) Cytolytic CD4 cells: direct mediators in infectious disease and malignancy
Cell. Immunol.
(2010)- et al.
CD107a as a functional marker for the identification of natural killer cell activity
J. Immunol. Methods
(2004)