Differentiation of anti-tumour cytotoxic T lymphocytes from autologous peripheral blood lymphocytes in non-Hodgkin’s lymphomas Laurence Chaperot, 1,2 Olivier Manches, 1,2 Jian Qing Mi, 1,2 Agnes Moine, 1 Marie-Christine Jacob, 1,2 Re ´my Gressin, 3 Jean-Paul Molens, 1,2 Jean-Jacques Sotto, 2,3 Dominique Leroux, 2 Jean-Claude Bensa 1,2 and Joe ¨l Plumas 1,2 1 R & D Laboratory, EFS Rho ˆne-Alpes, 2 Research Group on Lymphomas, Unite ´ UPRES 2021, and 3 CHU Michallon, Grenoble, France Received 17 April 2002; accepted for publication 31 May 2002 Summary. We have previously reported that specific anti- tumour cytotoxic T cells (CTL) can be differentiated from tumour-infiltrating lymphocytes (TIL) in non-Hodgkin’s lymphoma. We found that the combination of interleukin (IL)-1, IL-2 and IL-12 was very efficient for expansion of CD8 + T-cell receptor (TCR)ab + T cells and for development of their ability to specifically lyse tumour cells. In this study, we investigated whether anti-tumour T cells could be gen- erated from the peripheral blood of patients using the cul- ture protocol developed for TIL. Autologous T cells and tumour B cells from five patients were included in this study. It was found that polyclonal anti-tumour cytotoxic effector cells were generated when cultured in the presence of IL-1b, IL-2 and IL-12. Interestingly, tumour cells were lysed by perforin/granzyme-mediated cytolysis and not by CD95-mediated apoptosis. By performing inhibition experi- ments, it was observed that both CD8 + and CD4 + T cells were responsible for the cytotoxic effect and that they were able to recognize malignant B cells by either a major histocompatibility complex (MHC)-restricted or MHC-non- restricted mechanism. Intriguingly, in addition to inter- feron-c and tumour necrosis factor-a, IL-10 was secreted continuously during culture. The source of patient T cells used for the generation of anti-tumour CTL should be based on the results obtained with peripheral blood lymphocytes and TIL. Keywords: lymphoma, CTL, cytotoxicity, PBL, cytokines. Recent progresses in anti-tumour immunology have shown that the immune system of cancer patients can mount an effective response if appropriately stimulated. This consti- tutes the prerequisite of clinical trials using vaccination strategy with tumour peptides (Rosenberg et al, 1998; Marchand et al, 1999) or dendritic cells pulsed with tumour antigens (Nestle et al, 1998; Murphy et al, 1999; Thurner et al, 1999). In B cell-derived non-Hodgkin’s lymphoma (NHL), the use of idiotype alone (Kwak et al, 1992; Nelson et al, 1996; Bendandi et al, 1999), or in combination with dendritic cells (Hsu et al, 1996), produced clinical and biological responses in a significant number of patients, thereby confirming that anti-tumour-specific T lymphocytes exist and that the patient’s immune system is still able to develop an efficient anti-tumour response, even after chemotherapy. In a recent study, we reported that anti- tumour-specific cytotoxic T lymphocytes (CTL) could be differentiated from autologous tumour-infiltrating lympho- cytes (TIL) by culturing them with tumour cells in the presence of interleukin (IL)-2, IL-1 and IL-12 (Chaperot et al, 1999a). These data confirmed that a T cell-mediated immune response could be generated against tumour B cells. Moreover, these results also confirmed that malignant NHL B cells retained their antigen-presenting cell capacity (Plumas et al, 1995) mainly due to their expression of human leucocyte antigen (HLA) class I and class II molecules (Chaperot et al, 2000) and high levels of CD80 and CD86 (Chaperot et al, 1999b), the two crucial co-stimulatory molecules for accessory functions. However, the addition of appropriate cytokines was needed to efficiently activate TIL. IL-12 is of interest as this cytokine is produced by dendritic cells upon ligation of CD40 and enables the polarization of T cells towards a Th1 phenotype (Macatonia et al, 1995), responsible for the cell-mediated immune response. Correspondence: Joe ¨l Plumas or Laurence Chaperot, R & D Laboratory, EFS Rho ˆne-Alpes, BP 35, F-38701 La Tronche Cedex, France. E-mail: joel.plumas@wanadoo.fr or laurence.chaperot@ efs.sante.fr British Journal of Haematology, 2002, 119, 425–431 Ó 2002 Blackwell Publishing Ltd 425