Adoptive cell therapy (ACT) requires the generation of highly avid tumour-antigen-reactive T cells. Tumour-specific T cells, derived from tumour-infiltrating lymphocytes (TILs), can be efficiently isolated ex vivo from melanoma lesions using high levels of interleukin-2 (IL-2). TILs are successively selected for their ability to secrete high levels of interferon-γ (IFNγ) when cultured with autologous or allogeneic MHC-matched tumour-cell lines. Alternatively, cell-mediated lysis has been used to identify tumour-reactive T cells for transfer. Highly avid, tumour-antigen-reactive T-cell populations selected for ACT are rapidly expanded (to up to 10¹¹ cells) using CD3-specific antibody, exogenously supplied IL-2 and irradiated allogeneic peripheral-blood mononuclear ‘feeder’ cells, and are validated for activity before transfer. Patients now receive systemic immunosuppression before the adoptive transfer of antitumour lymphocytes. Published lymphodepleting regimens consist of a non-myeloablative, but lymphodepleting, conditioning chemotherapy comprised of cyclophosphamide and fludarabine before administration of T cells. Newer, as yet unpublished, regimens also include total body irradiation. ELISA, enzyme-linked immunosorbent assay. This figure is reproduced with permission from REF. 12 © (2005) Elsevier Science.

Computed tomography (CT) scans of the liver in a patient with metastatic melanoma show dramatic tumour regression of liver metastases after the administration of tumour-reactive tumour-infiltrating lymphocytes (TILs) following lymphodepletion. The patient is still disease-free after 27 months.

A | Adoptive cell therapy (ACT) in a lymphoreplete host. In a lymphoreplete environment, antitumour responses mediated by adoptively transferred tumour-reactive CD8⁺ T cells might be reduced because of: a | competition for antigen at the surface of antigen-presenting cells (APCs) and inefficient lymphocyte activation in the absence of co-stimulatory molecules by immature dendritic cells (DCs); b | reduced availability of activating cytokines (including interleukin-2 (IL-2), IL-7 and IL-15) by cellular ‘sinks’ for these cytokines, which include B cells, T cells and natural killer (NK) cells; and c | the suppressive activities of regulatory T (T_Reg) cells, myeloid suppressor cells (MSCs) and possibly NK cells. T_Reg-cell suppression is mediated by direct T-cell contact and possibly by the release of inhibitory cytokines such as IL-10 and transforming growth factor-β. MSCs mediate T-cell inhibition through direct T-cell contact and the use of enzymes involved in L-arginine metabolism such as the inducible forms of arginase and nitric-oxide synthase, ARG1 and NOS2. B | Systemic chemotherapy or radiation before ACT might modify the tumour-bearing host. APCs are reduced in number by direct killing but there might be a net increase in lymphocyte activation because of reduced competition for antigen at the APC surfaces. At the same time, as a result of the liberation of Toll-like receptor (TLR) agonists after mucosal damage, DCs might be mature, increasing lymphocyte activation. Activating cytokines, such as IL-2, IL-7 and IL-15 might be increased because of the removal of cellular ‘sinks’; and T_Reg cells, MSCs, NK cells and their suppressive activities are decreased. These modifications might promote the full activation of adoptively transferred tumour-reactive CD8⁺ T cells and ultimately tumour destruction.

At increasing strength of stimulation, naive CD8⁺ T cells proliferate and progressively differentiate through early, intermediate and late effector stages. The phenotypic and functional changes that characterize this process are illustrated as no expression (–), intermediate expression (+) and high expression (hi) of the various markers. T cells progressively lose telomere length and proliferative potential, and subsequently become senescent and undergo apoptosis. The progressive acquisition of full effector functions (dashed burgundy line) is associated with a decreased ability of T cells to cause tumour regression after adoptive transfer (black line). The molecular mechanisms underlying this inverse correlation might be comprised of: decreased expression by T cells of lymph-node homing and co-stimulatory molecules, which reduce activation of T cells in vivo; the inability of terminally differentiated T cells to produce interleukin-2 (IL-2); a reduction in the amount of receptors required to receive activating signals from homeostatic cytokines; and finally, an inversion of the expression of pro- and anti-apoptotic molecules with the corresponding acquisition of replicative senescence. Adoptively transferred tumour-infiltrating lymphocytes (TILs) contain several clonotypes with a differentiation state ranging between intermediate and late effector stages, whereas tumour-reactive CD8⁺ T-cell clones are uniformly late effector T cells. KLRG1, killer-cell lectin-like receptor G1. This figure is reproduced with permission from REF. 65 © (2005) Highwire Press.

a | Retroviral transduction of peripheral-blood lymphocytes (PBLs). PBLs at different stages of differentiation, naive (grey), early (green), intermediate (beige) and late effector (burgundy) are activated in vitro with CD3-specific antibody in the presence of interleukin-2 (IL-2) to promote integration of tumour-specific T-cell receptor (TCR) retroviral constructs. This procedure results in the generation of more-differentiated TCR transductants. Pairing with endogenous receptor can reduce the number of tumour-specific TCRs. b | Lentiviral transduction of naive CD8⁺ T cells. Naive CD8⁺ T cells isolated through selective sorting can be transduced with tumour-specific TCR by using lentiviral constructs that do not require activation and consequent differentiation. Pairing with endogenous receptor can reduce the number of tumour-specific TCRs. c | Lentiviral transduction of haematopoietic stem cells (HSCs). CD34⁺CD38⁻ HSCs isolated though selective sorting can be transduced with tumour-specific TCR using lentiviral constructs. HSCs can be induced to differentiate into naive CD8⁺ T cells in vitro through Notch-mediated signalling. Repression of recombination-activating genes by the transduced tumour-specific TCR allows for the uniform expression of tumour-specific TCRs.