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Immune Checkpoint Antibodies for Flow Cytometry, IHC, and Functional Bioassays
Harness immune checkpoints to combat tumors
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As critical members of the adaptive immune system, T cells are capable of mounting an efficient immune response against tumor cells. As T cells circulate in the body, they are constantly interrogating proteins on the surface of cells they encounter. If a cell is identified as “foreign”, the T cells will unleash an attack against the invading cell. Tumor cells, however, can evade detection by masking their surface with proteins of normal cells. These proteins—referred to as immune checkpoints—can affect immunoregulatory pathways by either boosting (co-stimulatory) or restricting (co-inhibitory) the immune responses of T cells [1,2]. So far, a great number of immune checkpoint proteins have been identified (Figure 1).
Figure 1. Multiple co-stimulatory and co-inhibitory receptor–ligand interactions between antigen-presenting cells (APCs) and T cells. T cell receptors (TCRs) detect antigens on the surface of APCs in the form of antigen-complexed major histocompatibility complexes (MHCs), and this antigen-specific recognition is necessary but not sufficient for an effective T cell response. For T cell activation or suppression, T cells must recognize their cognate antigens through TCRs and then respond to co-stimulatory (for activation) or co-inhibitory (for suppression) receptor–ligand interactions, examples of which are shown in this schematic. One important family of membrane-bound molecules that bind both co-stimulatory and co-inhibitory receptors is the B7-CD28 family shown in purple boxes; all of the B7 family members and their known ligands belong to the immunoglobulin superfamily. Another major category of signals arise from tumor necrosis factor (TNF) family members (shown in green boxes), which regulate the activation of T cells in response to cytokines.
Antibodies for dissecting the immune checkpoint pathways
Antibodies that target immune checkpoint pathways—such as PD-1/PD-L1 (Figure 2), CTLA4/CD80 and CD86, LAG3/MHC II, and TIM3/GAL9—have proven effective as immunotherapeutic agents in cancer treatment. For example, PD-1/PD-L1 is a co-inhibitory pathway that functions to restrict the immune system by constraining T cell anti-tumor activity. Studies with antibodies (e.g., nivolumab and pembrolizumab) that target the PD-1/PD-L1 pathway and suppress its co-inhibitory function have shown durable clinical responses in combating certain cancers, even in patients with advanced-stage cancer [1,2].
Recently, the co-inhibitory receptor TIGIT has been found to be expressed on the surface of a variety of lymphocytes, including effector and regulatory CD4+ T cells, follicular helper CD4+ T cells, effector CD8+ T cells, natural killer (NK) cells, and memory T cells (Figure 3). The expression of TIGIT is markedly enriched on tumor-infiltrated T cells [3]. In addition, the TIGIT+ regulatory T cells in tumor tissue co-express TIM3, and TIM3 and TIGIT work together to suppress anti-tumor immunity [3]. Similarly, therapeutic agonists of DR3, a co-stimulatory receptor that is expressed primarily in tissues enriched in lymphocytes (Figure 4), can also be used to stimulate regulatory T cell expansion, which can reduce inflammation and enable patients to produce an effective anti-tumor response [4]. These examples show that releasing the anti-tumor immune response by blocking co-inhibitory immune checkpoints or activating co-stimulatory immune checkpoints is a promising approach for triggering anti-tumor responses and mediating durable cancer regressions.
Figure 2. The blockade of immune checkpoint PD-1/PD-L1 in immunotherapy. This figure shows PD-1 and PD-L1 for illustrative purposes, although the concept likely applies to multiple immune checkpoints. (A) When PD-1 on the T cell surface binds its ligand PD-L1 on the tumor cell, the T cell becomes inactivated, leaving the tumor cell intact and growing. Blocking the activity of PD-1 or PD-L1 with inhibitors (e.g., antibodies) can prevent the interaction of PD-1 and PD-L1, enabling the T cell to stay active and launch an anti-tumor immune response to the tumor cell, releasing inflammatory signals such as IFN-gamma. (B) Immunohistochemical staining of human brain tissue was performed using an anti-PD1 (CD279) polyclonal antibody (25 μg/mL). (C, D) Unstimulated or PHA-stimulated normal human peripheral blood cells have been stained with PD-1 or PD-L1 antibody as follows, and viable cells in the lymphocyte gate were detected using Invitrogen eBioscience Fixable Viability Dye eFluor 520. PD-1 or PD-L1 antibodies include: (C)APC mouse IgG1 kappa isotype control (blue histogram) or APC anti–human CD279 (PD-1) antibody (purple histogram). (D)APC mouse IgG1 kappa isotype control (blue histogram) or APC anti–human CD274 (PD-L1, B7-H1) antibody (purple histogram).
Figure 3. Expression of TIGIT on CD4+ CD45RO+ T cells. Normal human peripheral blood cells were stained with APC anti–human CD4 antibody (clone RPA-T4, Cat. No. 17-0049-42), PerCP-eFluor 710 anti–human CD45RO antibody (clone UCHL1, Cat. No. 46-0457-42), and PE mouse IgG1 kappa isotype control (top, Cat. No. 12-4714-82) or PE anti–human TIGIT antibody (bottom, Cat. No. 12-9500-42). CD4+ cells in the lymphocyte gate were used for analysis.
Figure 4. Expression of DR3 in Ramos cells. Immunocytochemical fluorescence analysis was performed on fixed and permeabilized Ramos cells for detection of (A) endogenous DR3 (TNFRSF25) using Invitrogen anti-DR3 (TNFRSF25) ABfinity recombinant rabbit monoclonal antibody (2 μg/mL) in conjunction with Invitrogen Alexa Fluor 488 goat anti–rabbit IgG Superclonal secondary antibody (green, 1:2000 dilution), (B) nuclei using Invitrogen SlowFade Gold Antifade Mountant with DAPI (blue), and (C) cytoskeletal F-actin using rhodamine phalloidin (red, 1:300 dilution). (D) The composite image shows staining of DR3 (TNFRSF25), nuclei, and actin. To assess background fluorescence, control cells were stained similarly except that no primary antibody was used (data not shown). The images were captured at 60x magnification on a Nikon™ Eclipse™ Ti-U
Check out our checkpoint antibodies
Despite recent breakthroughs in immunotherapy to treat melanoma of the skin, non-small cell lung cancer, kidney and bladder cancers, head and neck cancers, and Hodgkin lymphoma, the complex biology of the immune checkpoint pathways is still far from being understood. We offer a wide range of antibodies designed to study immune checkpoints (Table 1), as well as blocking antibodies, proteins, assays, and more. Our experienced custom service team can also save you time by developing antibodies to meet your specifications.
References
Table 1. Selected antibodies for flow cytometry, immunohistochemistry (IHC)/immunofluorescence (IF), and functional bioassays.
| Immune checkpoint | Anti-human antibody (Cat. No.) | Anti-mouse antibody (Cat. No.) | ||||
|---|---|---|---|---|---|---|
| Flow cytometry | IHC/IF | Functional bioassay | Flow cytometry | IHC/IF | Functional bioassay | |
| B7-H3 (CD276) | MA1-74441, 46-2769-42 | MA5-15693, MA1-74441 | 16-5937-85 | 16-5973-81 | 16-5973-81 | |
| B7-H4 (VTCN1) | MA5-16845, 17-5949-42 | MA1-74439, MA1-74440 | 16-5949-82 | 12-5970-83, 12-5972-82 | 16-5972-81 | |
| B7-H5 (VISTA) | PA5-52493 | 46-5919-82 | PA5-47032 | 16-5919-85 | ||
| BTLA (CD272) | MA5-16843, 13-5979-80 | PA5-22248, MA1-74212 | 16-5979-82 | 12-5950-82, 17-5956-82 | 16-5950-82 | |
| CD27 | 11-0279-42, 11-0271-85 | 16-0272-85, PA5-28036 | 16-0271-85 | MA5-17902, MA5-17904 | 12-0271-83 | 16-0272-85 |
| CD28 | 61-0289-42, MA1-10170 | MA1-10166, MA5-17005 | 16-0281-86 | MA1-34061, 14-0281-86 | MA1-10172 | 16-0289-85 |
| CD30 | MA5-12632, 11-0309-42 | MA5-12632 | 12-0301-82 | MA1-90234 | 16-0301-85 | |
| CD30L (CD153) | MA5-23859 | PA5-47045 | MA5-23859 | A18356, 12-1531-83 | 14-1531-85 | 14-1531-85 |
| CD40 | MA1-80926, 11-0409-42 | 14-0409-82, 700121 | 16-0409-85 | MA5-17853, 46-0401-82 | 14-0401-86 | 16-0402-86 |
| CD40L (CD154) | MA1-33895, 11-1548-42 | PA5-13483 | 16-1548-82 | HMCD15401, 46-1541-82 | MA1-10108 | 16-1541-85 |
| CD70 | MA5-17727, 50-0709-42 | PA5-32701, MA5-17726 | 14-0701-85, MA1-81645 | 16-0701-85 | ||
| CD80 | MA1-19215, 11-0809-42 | MA5-15512, MA1-90872 | 16-0809-85 | MA1-70090, 11-0801-86 | 14-0801-85 | 16-0801-85 |
| CD86 | MA1-81186, 12-0869-42 | MA1-12172, MA1-10293 | 14-0869-82 | 12-0861-83, 11-0862-85 | 14-0862-85 | 16-0861-85 |
| CD96 (Tactile) | 46-0969-42, MA5-24280 | PA5-64286 | MA5-24281, 12-0960-80 | 16-0960-82 | ||
| CD112 (Nectin-2) | MA1-35890, 17-1128-42 | PA5-29757 | ||||
| CD134 (OX40) | MA5-23591, 11-1347-42 | PA5-34516, 14-1347-82 | 12-1341-83, MA1-70087 | 14-1341-85, MA5-17916 | 16-1341-85 | |
| CD137 (4-1BB) | MA5-13739, 11-1379-42 | MA5-13739 | PA5-47037 | PA5-47967, 25-1371-82 | 16-1371-85 | |
| CD137L (4-1BB L) | PA5-47291 | 46-5901-82 | ||||
| CD152 (CTLA-4) | 12-1528-42, 25-1529-42 | PA5-23967, PA5-47547 | 16-1529-82 | 61-1522-82, HMCD15201 | 16-1521-85 | |
| CD155 (PVR) | MA5-13493, 46-1550-42 | MA5-13490 | 17-1551-82 | MA5-24315 | ||
| CD223 (LAG3) | 46-2239-42 | MA5-24284, 11-2231-82 | 16-2231-85 | |||
| CD226 (DNAM1) | PA5-38444, PA5-31111 | MA5-17990, 16-2261-85 | PA5-36393 | 16-2261-85 | ||
| CD252 (OX40L) | PA5-24552 | PA5-34516 | 46-5905-82 | |||
| CD258 (LIGHT) | 46-2589-42, 17-2589-42 | |||||
| CD273 (PD-L2) | 17-5888-42, MA5-16839 | PA5-20344 | 16-5888-82 | 12-5986-83, 11-9972-85 | 14-5986-85 | 16-5986-82 |
| CD274 (PD-L1) | 46-5983-42, MA5-16840 | 14-5983-82 | 16-5983-82 | 62-5982-80, 13-9971-81 | 14-5982-85 | 16-5982-85 |
| CD278 (ICOS) | 11-9948-42, MA5-23680 | 14-9948-82 | 16-9948-82 | 46-9940-82, 11-9942-82 | 14-9949-82 | 16-9942-85 |
| CD279 (PD-1) | 12-9969-42, 12-2799-42 | 14-2799-80, 14-9969-82 | 16-9989-38 | 11-9981-82, 25-9985-82 | 14-9985-85 | 16-9985-85 |
| CD357 (GITR) | 48-5875-42, MA5-23854 | PA5-46810, PA5-47883 | PA5-47883 | MA5-17934, 11-5874-82 | 16-5874-83 | 16-5874-83 |
| DR3 (TNFRSF25) | 702277, 12-6603-42 | PA1-30533, PA5-28293 | 711309, MA5-23838 | 711309 | ||
| Galectin-9 | 50-9116-42 | PA5-32252, PA5-50966 | 16-9116-85 | PA5-47503 | MA5-24369 | |
| GITRL | PA5-20161 | MA5-23852, PA5-47885 | PA5-20161 | |||
| HVEM | MA1-25958, 12-5969-80 | PA5-20236, PA5-26103 | 17-5962-82 | PA5-20237 | 16-5962-85 | |
| ICOSL (B7RP1, B7-H2) | MA1-17756, 12-5889-42 | 13-5889-82 | 16-5889-82 | 50-5985-82, MA5-24270 | PA5-47161 | 16-5985-85 |
| IDO | MA5-23595, 12-9477-42 | PA5-29819, PA5-12305 | 46-9473-82 | PA5-24598 | ||
| TIGIT | 46-9500-42 | 16-9500-85 | 12-9501-82 | 16-9501-85 | ||
| TIM3 | 47-3109-42 | 16-3109-85 | 16-5871-85, MA5-1795 | 14-5871-85 | 16-5871-85 | |
| TL1A | 46-7911-82 | 46-7911-82 | ||||
| VSIG4 | 17-5757-42 | PA5-52018 | 17-5752-82 | |||
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