Expression of costimulatory molecules CD80 and CD86 and their receptors CD28, CTLA-4 on malignant ascites CD3+ tumour-infiltrating lymphocytes (TIL) from patients with ovarian and other types of peritoneal carcinomatosis

Abstract

Costimulation of T lymphocytes by the leucocyte surface molecules CD80 and CD86 expressed on antigen-presenting cells (APC) is required for the development of T cell responses. The CD28 and CTLA-4 molecules on T cells serve as receptors for the CD80 and CD86 costimulatory antigens. We have examined the frequency of expression of CD80 (B7.1), CD86 (B7.2), CD28 and CTLA-4 surface antigens on TIL isolated from malignant ascites or peritoneal washings of 26 patients with ovarian carcinoma and five patients with non-ovarian peritoneal carcinomatosis. Expression of CD80 and CD86 antigen was detected by reverse transcription-polymerase chain reaction (RT-PCR), and by FACS analysis. Significantly higher proportions of intraperitoneal CD3+ cells expressed CD86 antigen than the CD80 antigen (14 +/- 9% versus 3 +/- 3%, P < 0.05). Moreover, CD3+CD86+ cells were significantly more frequent in the peritoneal fluid (14 +/- 9%) than in the peripheral blood (3 +/- 0.4%, P < 0.05) of ovarian patients or normal controls (3 +/- 1%). CTLA-4 and CD28 antigen were expressed, respectively, on 9 +/- 4% and 86 +/- 14% of ascitic CD3+ cells of ovarian cancer patients. Both CD80 and CD86 antigens were expressed primarily on HLA-DR+ ascites TIL and were present in a very low proportion of HLA-DR- ascites TIL. These HLA-DR+ cells may represent a population of lymphocytes that have been activated in vivo, and function as APC. An anti-CD86 MoAb or a combination of anti-CD86 and anti-CD80 MoAbs significantly inhibited the proliferation of cultured intraperitoneal TIL. We have shown that in addition to CD28 and CTLA-4, CD3+ intraperitoneal TIL express the costimulatory molecules CD80 and CD86. The expression of these molecules on T cells could be dependent upon certain factors in the tumour microenvironment that could determine the outcome of in vivo immune responses.

Fig. 1

Presence of the transcripts for CD86 and CD80 in nylon wool-separated intraperitoneal TIL. The intraperitoneal TIL were isolated from ascitic fluid by nylon wool extraction. RNA was isolated and CD80 and CD86 transcripts were studied by reverse transcription-polymerase chain reaction (RT-PCR), as described in Patients and Methods. The results shown are from three patients. Lanes 1–3, CD86 (lane 1, patient C28; lane 2, C30; lane 3, C29); lanes 4–6, CD80 (lane 4, C28; lane 5, C30; lane 6, C29); lanes 7–9, β-actin (lane 7, C28; lane 8, 30; lane 9, 29).

Fig. 2

Abrogation of phytohaemagglutinin (PHA)-stimulated proliferation of cultured intraperitoneal TIL. Nylon wool-separated lymphocytes from patient C28 were suspended in RPMI supplemented with 10% fetal calf serum (FCS), granulocyte-macrophage colony-stimulating factor (50 ng/ml) and tumour necrosis factor-alpha (50 ng/ml). After 3 days the culture medium was replaced with RPMI 10% FCS and 600 U/ml IL-2. After 4 weeks the cells (99% CD3⁺, 35% CD80, 45% CD86, 24% CTLA-4, and 92% CD28) were harvested and incubated with PHA and/or respective antibodies. Results shown are representative of three experiments.