T cells require different signals for appropriate expansion and acquisition tumoricidal functions . Signal 1 is initiated after cognate T-cell receptor binding to a MHC-peptide complex on the surface of an antigen-presenting cell. Co-regulatory molecules including CD28 and CTLA-4 that bind to CD80 and CD86 deliver or modulate signal 2. Moreover, other co-regulatory interactions and paracrine cytokines, both stimulatory and inhibitory, shape and direct the type of response (signal 3). Crucially, negative regulators modulate the grade of T cell activation and protect the organism from deleterious effects mediated by uncontrolled T cell activation. CTLA-4 is one of these co-inihibitory molecules expressed on activated T cells. It binds CD80 and CD86 with an affinity at least 100-fold higher than CD28. A fully human mAb blocking this interaction has been approved for the treatment of metastatic melanoma [5, 6].
PD-1 is a co-inhibitory receptor that shows homology with CD28 and whose expression is induced following activation on CD4+ and CD8+ T cells as well as on other immune cells. Its cognate ligands are the members of the B7 family PD-L1 (B7-H1) and PD-L2 (B7-DC). PD-1 triggering on the T cell leads to a non pro-apoptotic inhibition of T cell proliferation and activation dependent on recruitment of a tyrosine phosphatase that represses signaling at immune synapses . In animal models of infectious disease, PD-1 is upregulated in both acute and chronic LCMV infection but stays high in chronic infection marking exhausted T cells. Indeed, PD-1 is considered as a decisive exhaustion marker along with LAG-3, CD160, BTLA, and 2B4. Three antibodies against PD-1 have started clinical trials in patients with advanced cancers. Early reports suggest excellent clinical activity with conspicuously long-lasting responses and disease stabilization [2, 29]. In addition there is interest in selectively modulating PDL1 given other inhibitory interactions of this molecule  and the anti-apoptotic signals of PDL1 to tumor cells .
HVEM is a member of the TNFR superfamily with multiple ligands, both stimulatory and inhibitory. Gordon Freeman’s group (Dana Farber Cancer Institute) has demonstrated that deletion of HVEM cysteine rich domain 1 (CRD1) interferes with the binding capacity of the inhibitory receptors BTLA and CD160. In contrast, HVEM lacking CRD1 is a stimulatory receptor through interactions with LIGHT. B16 melanoma expressing HΔD1 (HVEM with deletion of CRD1 domain) becomes immunogenic and mice harboring irradiated B16-HΔD1 survive longer compared to B16 recipient mice. Treatment efficacy is further improved with the concomitant use of anti-CTLA4 and anti-PD-1 mAbs. The combined strategy greatly enhances lymphocyte trafficking into tumor sites. Optimal immunotherapy may involve a combination of blockade of inhibitory signals and provision of costimulatory signals as if releasing the brakes and stepping on the gas pedal of a car . The above strategy may be used to endow cellular vaccines with immune stimulating properties, through genetically introduced expression of molecules such as GM-CSF, Flt-3-ligand, ICOS-ligand and others [32, 33].
CD40 is a costimulatory molecule expressed on antigen presenting cells and frequently on neoplastic cells with no intrinsic kinase activity. CD40-CD40L interactions facilitate dendritic cells (DCs) to increase expression of CD80 and CD86 and threby activate T cells. A fully human mAb (CP-870,893) that recognizes CD40 has been developed and shown to activate human DCs. This mAb is currently in clinical trials for several types of malignant tumors. Robert Vonderheide (University of Pennsylvania) described his experience with anti-CD40 mAb in pancreatic cancer . Agonist CD40 mAb combined with gemcitabine induced major clinical remissions in patients with pancreatic adenocarcinoma. CD40-dependent treatment effects can be reproduced in a genetically engineered mouse model of the disease. Strikingly, the therapeutic mechanism fol-lowing CD40 activation is not T cell-dependent, but rather is macrophage-dependent. Intriguingly, CD40-activated macrophages rapidly infiltrate and kill tumor cells, and facilitate depletion of tumor stroma. CD40 can work effectively to induce tumor infiltrating T cells but requires gemcitabine for antigen priming outside the tumor microenvironment.
Monoclonal antibodies that bind stimulatory or inhibitory receptors are excellent pharmacological tools to magnify the role of receptor-ligand pairs in cancer immunotherapy. However, there are some unknowns about mechanisms underlying the action of these mAbs. Martin Glennie (University of Southampton) discussed the physiology of Fc receptors and effects on the function of anti-CD40 mAb . This British group concomitantly with Li and Ravetch  at Rockefeller University (NYC) found that the isotype of the mAb plays a crucial role in the activity of anti-CD40 mAb. IgG1 is optimal in mouse models due to its ability to bind FcγRIIB in vivo, thereby allowing cross-linking of the FcR receptor upon cell-to-cell contact with the responding leukocytes. Early results from the same group suggest that other immunostimulatory mAbs are FcR cross-linking dependent. Understanding these mechanisms will allow fine-tuning of agonistic mAbs and may optimize the benefit:toxicity ratio of these agents.
The immune system recognizes CpG oligonucleotides through TLR9 receptors that activate B-lymphocytes and DCs. Intratumoral injection of CpG after radiation therapy promotes systemic lymphoma eradication in mice  with interesting results in humans . CpG in situ vaccination induces anti-tumor T cells that express CD45RO and CD137. This type of treatment is practical (no need for a customized product), safe and repeatable. The group of Ron Levy (Stanford University) identified combinations with immunostimulatory mAbs that extend the efficacy of CpG intratumoral injections in murine models. In transplanted tumors, OX40 and CTLA4 were highly expressed on tumor-infiltrating lymphocytes compared to T cells in the spleen or draining lymph nodes. Most OX40/CTLA4+ T cells infiltrating tumors were FOXP3+ regulatory T cells. When mice were intratumorally treated with anti-OX40 and anti-CTLA4 mAbs, FOXP3 + CD4+ T cells were depleted and established tumors eliminated using as low as 1/100 of the needed dose when given systemically. This intratumoral route was also capable of inducing the rejection CNS leukemia metastases. The message is: “treat locally, cure systemically”.