Dysregulated expression of both the costimulatory CD28 and inhibitory CTLA-4 molecules in PB T cells of advanced cervical cancer patients suggests systemic immunosuppression related to disease progression

Abstract

Cervical cancer (CC) occurs more frequently in women who are immunosuppressed, suggesting that both local and systemic immune abnormalities may be involved in the evolution of the disease. Costimulatory CD28 and inhibitory CTLA-4 molecules expressed in T cells play a key role in the balanced immune responses. There has been demonstrated a relation between CD28, CTLA-4, and IFN genes in susceptibility to CC, suggesting their importance in CC development. Therefore, we assessed the pattern of CD28 and CTLA-4 expression in T cells from PB of CC patients with advanced CC (stages III and IV according to FIGO) compared to controls. We also examined the ability of PBMCs to secrete IFN-gamma. We found lower frequencies of freshly isolated and ex vivo stimulated CD4 + CD28+ and CD8 + CD28+ T cells in CC patients than in controls. Loss of CD28 expression was more pronounced in the CD8+ T subset. Markedly increased proportions of CTLA-4+ T cells in CC patients before and after culture compared to controls were also observed. In addition, patients' T cells exhibited abnormal kinetics of surface CTLA-4 expression, with the peak at 24 h of stimulation, which was in contrast to corresponding normal T cells, revealing maximum CTLA-4 expression at 72 h of stimulation. Of note, markedly higher IFN-gamma concentrations were shown in supernatants of stimulated PBMCs from CC patients.

Conclusions: Our report shows the dysregulated CD28 and CTLA-4 expression in PB T cells of CC patients, which may lead to impaired function of these lymphocytes and systemic immunosuppression related to disease progression.

Fig. 1

CD28 expression in peripheral blood CD8+ T cells from patients with CC and from healthy controls before and after 24 h, 48 h and 72 h stimulation with anti-CD3 MoAb + rIL-2. The dot plots and histograms show representative data, illustrating the analysis method for identification of CD3+/CD8+ cells expressing CD28 following three-color staining. (a) The dot plots show the forward scatter/side scatter (FSC/SSC) distribution and the gate (region R1) was used to select lymphocytes for analysis. The R1 gated events were then analyzed for CD3/PerCP and CD8/FITC staining and double-positive cells (CD3 + CD8+) were gated (region R2). The dot plots show representative data from one patient with CC. (b) The final histograms. The double-gated populations were then analyzed for CD28/RPE. The gray histograms represent the isotype controls. The numbers located on the histograms represent the percentage of CD3 + CD8+ cells expressing CD28 molecule

Fig. 2

The mean percentage of peripheral blood CD4 + CD28+ T cells (a) and CD8 + CD28+ T cells (b) before and after ex vivo 24, 48 and 72 h of anti-CD3 + rIL-2 stimulation in CC patients and healthy controls

Fig. 3

Surface CTLA-4 expression in peripheral blood CD3+ cells from patients with CC and from healthy controls before and after 24 h, 48 h and 72 h stimulation with anti-CD3 MoAb + rIL-2. The dot plots and histograms show representative data, illustrating the analysis method for identification of CD3+ cells expressing CTLA-4 molecule following double-color staining. (a) The dot plots show the forward scatter/side scatter (FSC/SSC) distribution and the gate (region R1) was used to select lymphocytes for analysis. The R1 gated events were then analyzed for CD3/FITC staining and CD3-positive cells were gated (region R2). The dot plots show representative data from one patient with CC. (b) The final histograms. The double-gated populations were then analyzed for CTLA-4 expression. The gray histograms represent the isotype controls. The numbers located on the histograms represent the percentage of CD3+ cells expressing surface CTLA-4

Fig. 4

The mean percentage of peripheral blood T cells expressing surface CTLA-4 (CD3 + sCTLA-4+ cells) (a) and cytoplasmic CTLA-4 (CD3 + cCTLA-4+ cells) (b) before and after ex vivo 24, 48 and 72 h of anti-CD3 + rIL-2 stimulation in CC patients and healthy controls