The immune system is a collection of cells, tissues, and organs in the body that defend against attacks by “foreign” invaders. Once the invader is recognized, the immune response is triggered. T cells become activated after recognizing major histocompatibility complex (MHC) molecules on antigen-presenting cells (APCs) with the T cell receptor, and co-signaling molecules. The immune system has its own immunoregulators, referred to as immune checkpoints. The immune checkpoint molecules have both positive and negative modulatory effects on T cell activation.

Immune checkpoint therapies, targeting regulatory pathways in T cells to enhance antitumor immunity, are the focus of research on the treatment of several forms of cancer. Due to the dynamic nature of the immune response and multifaceted regulation of immune signaling pathways, immunity can overshoot its targets and attack healthy tissues or organs after release from checkpoint arrest. The way forward for immune checkpoint therapy lies in better understanding of the interaction between the immune system and tumor cells.

Provided below are overviews of some of the key immune checkpoint research targets to consider.


T cell activation is generally self-limited, as activated T cells express receptors such as PD-1 (also known as PDCD-1) that mediate inhibitory signals from the antigen-presenting cells (APC). PD-1 can bind to two different but related ligands, PD-L1 and PD-L2. Upon binding to either of these ligands, signals generated by PD-1 decrease TCR-mediated proliferation and cytokine production. Increased PDL-1 expression is associated with many murine and human cancers and can be further up-regulated upon IFN-gamma stimulation. Thus, PD-L1 might play an important role in immune evasion by tumors.

Flow cytometry analysis of PD-1

Flow cytometry analysis of PD-1 and B7-H1. C57BL/6 splenocytes were stimulated with ConA and then stained with APC anti–mouse CD3e (Cat. No. A18605) and 0.5 µg PE anti–mouse CD279 (PD-1) (Cat. No. A18700) (upper right panel) or 0.5 µg PE Armenian hamster IgG isotype control (upper left panel). Staining of C57BL/6 splenocytes with APC anti–mouse CD3e and PE rat IgG2a kappa isotype control (bottom left panel) or PE anti–mouse CD274 (B7-H1) (Cat. No. A14764) (bottom right panel). Cells in the lymphocyte gate were used for analysis.


DR3/TNFRSF25 is a member of the TNF receptor superfamily. This receptor is expressed preferentially in tissues enriched in lymphocytes, and it may play a role in regulating lymphocyte homeostasis. The sole known ligand for DR3 is the TNF superfamily member TL1A (TNF like cytokine 1A, TNFSF15). The signal transduction of this receptor is mediated by various death domain–containing adaptor proteins. Knockout studies in mice have suggested the role of DR3/TNFRSF25 in the removal of self-reactive T cells in the thymus. Studies have also suggested a role for TL1A and DR3 in various human diseases and animal disease models, including rheumatoid arthritis (RA), collagen-induced arthritis (CIA), and inflammatory bowel disease (IBD).

Immunofluorescence analysis of DR3

Immunofluorescence analysis of DR3/TNFRSF25. The experiment was performed on fixed and permeabilized Ramos cells for detection of endogenous DR3/TNFRSF25 using ABfinity Anti-DR3/TNFRSF25 Recombinant Rabbit Monoclonal Antibody (Cat. No. 702277, 2 µg/mL) and labeled with Goat Anti–Rabbit IgG (H+L) Superclonal Secondary Antibody, Alexa Fluor 488 conjugate (Cat. No. A27034, 1:2,000). Panel a shows representative cells that were stained for detection and localization of DR3/TNFRSF25 protein (green); panel b is stained for nuclei (blue) using SlowFade Gold Antifade Mountant with DAPI (Cat. No. S36938); panel c represents cytoskeletal F-actin staining using Alexa Fluor 555 Rhodamine Phalloidin (Cat. No. R415, 1:300); panel d is a composite image of panels a, b, and c clearly demonstrating localization of DR3/TNFRSF25 in the membrane. The images were captured at 60x magnification.


T cell immunoglobin mucin-3 (TIM-3) is expressed on the surface of human myeloid leukemia stem cells in many types of human acute myeloid leukemia (AML), but not on hematopoietic stem cells (HSCs). TIM-3 is overexpressed in Hodgkin's disease tissue and might participate in the interaction between the Hodgkin and Reed-Sternberg (H&RS) cells with their surrounding cells. Galectins are a family of beta-galactoside–binding proteins implicated in modulating cell–cell and cell–matrix interactions. Galectin-9 (Gal-9) is the ligand of TIM-3; together these proteins constitute an autocrine loop critical for leukemic stem cell (LSC) self-renewal and development of human AML and thus might play a role in the pathogenesis of this disease and/or its associated immunodeficiency.

Immunohistochemical analysis of Galectin-9

Immunohistochemical analysis of Galectin-9 was detected in perfusion fixed frozen sections of mouse colon using Rat Anti-mouse Galectin-9 Monoclonal Antibody (Cat. No. MA5-24369) at 25 µg/mL overnight at 4 °C. Tissue was stained using the Anti-Rat HRP-DAB (brown and counterstained with hematoxylin (blue). Specific staining was localized to plasma membranes and cytoplasm of epithelial cells.


CTLA4 is a member of the immunoglobulin superfamily and encodes a protein that transmits an inhibitory signal to T cells. Mutations in this gene have been associated with insulin-dependent diabetes mellitus, Graves’ disease, Hashimoto thyroiditis, celiac disease, systemic lupus erythematosus, thyroid-associated orbitopathy, and other autoimmune diseases. CD80 (B7-1) and CD86 (B7-2) are ligands of T cell critical costimulatory molecule CD28 and of an inhibitory receptor CTLA-4 (CD152). Both B7 molecules are expressed on professional antigen-presenting cells and are essential for T cell activation. Differences in CD80 and CD86 competency have not been fully elucidated yet; results from existing studies conflict about their respective roles in initiating or sustaining the T cell immune response.

Immunocytochemical analysis of CTLA-4

Immunocytochemical analysis of CTLA-4 was detected in immersion fixed human peripheral blood mononuclear cells treated with treated with PMA and calcium ionomycin using Goat Anti-human CTLA-4 Antigen Affinity-purified Polyclonal Antibody (Cat. No. PA5-47547) at 15 µg/mL for 3 hours at room temperature. Cells were stained using the 557-conjugated Anti-Goat IgG Secondary Antibody (re and counterstained with DAPI (blue). Specific staining was localized to cell surfaces.


TIGIT, a co-inhibitory receptor, is expressed on the surface of a variety of lymphoid cells, especially on Foxp3+ regulatory T cells. The expression of TIGIT is markedly enriched on tumor-infiltrating T cells. The poliovirus receptor (PVR, CD155), a member of the Ig superfamily with 3 Ig domains in the arrangement V-C-C, is the high-affinity receptor for TIGIT. Binding with its receptor CD155, TIGIT causes increased secretion of IL-10 and decreased secretion of IL-12B and suppresses T cell activation by promoting the generation of mature immunoregulatory dendritic cells.

Immunofluorescent analysis of CD155 (green) showing staining in the membrane of Hela cells (right) compared to a negative control without primary antibody (left). Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with a CD155 monoclonal antibody (Cat. No. MA5-13493) in 3% BSA-PBS at a dilution of 1:20 and incubated overnight at 4 °C in a humidified chamber. Cells were washed with PBST and incubated with a DyLight-conjugated secondary antibody in PBS at room temperature in the dark. F-actin (red) was stained with a fluorescent red phalloidin and nuclei (blue) were stained with Hoechst or DAPI. Images were taken at a magnification of 60x.


LAG3 (CD223) is a cell surface molecule expressed on activated T cells, NK cells, B cells, and plasmacytoid dendritic cells. Binding with its major ligand, Class II MHC, LAG3 plays a role in modulating dendritic cell function. Based on research studies, it is also extensively co-expressed with PD-1 on tumor-infiltrating CD4+ and CD8+ T cells in several transplantable tumors.


VISTA (B7-H5, PD-1H or C10orf54) is an orphan receptor expressed on T cells, myeloid cells, and NK-cells. It functions as a negative regulator of T-cell immunity. The receptor for VISTA remains unknown. Studies in mice show VISTA inhibits T-cell activation and proliferation and induces regulatory T cell differentiation. VISTA and PD-1 suppress T-cell function in a synergistic manner indicating the possibility of combined therapy targeting both VISTA and PD-1 to enhance anti-tumor immunity.

Flow cytometric analysis of human peripheral blood mononuclear cells (PBMCs, upper panel) and mouse splenocytes (bottom panel). Normal human peripheral blood cells were stained with CD14 Monoclonal Antibody, PerCP-eFluor 710 (Cat. No. 46-0149-42) and Mouse IgG1 kappa Isotype Control, PE (Cat. No. 12-4714-82) (upper left) or VISTA Monoclonal Antibody, PE (Cat. No. 12-1088-42) (upper right). Mouse splenocytes were stained with CD8a Monoclonal Antibody, FITC (Cat. No. 11-0081-82) and 0.5 µg of Rat IgG2a kappa Isotype Control, Super Bright 436 (Cat. No. 62-4321-82) (bottom left) or 0.5 µg of VISTA Monoclonal Antibody, Super Bright 436 (Cat. No. 62-1083-82) (bottom right). Cells in the lymphocyte gate were used for analysis. Quadrant markers were set based on control antibody staining.