With the advancement of immunotherapeutics, the urge to understand the tumor microenvironment has never been more pressing. Immunohistochemistry is a powerful tool used to examine protein expression, distribution, and activation in situ.

An Introduction to Multiplex Immunohistochemistry

With the advancement of immunotherapeutics, the urge to understand the tumor microenvironment has never been more pressing. Immunohistochemistry is a powerful tool used to examine protein expression, distribution, and activation in situ. Antibodies specific to an antigen of interest are used to detect the antigen in thin sections of flash frozen or formalin-fixed paraffin-embedded tissue. Visualization of the antigen is achieved using either an enzymatic reaction that induces chromogen precipitation at the site of antibody-antigen binding, or fluorescent reporters. Fluorescent reporters may be directly conjugated to the primary antibody used to detect the antigen of interest (direct immunofluorescence), or may be attached to a secondary antibody that detects the species-specific primary antibody (indirect immunofluorescence). The latter is more common, as it achieves more sensitive antigen detection.

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Simultaneous detection of multiple distinct proteins of interest within a single sample can be achieved with carefully optimized fluorescent multiplex immunohistochemistry (mIHC) using tyramide signal amplification (TSA).

Practical Overview of Multiplex Immunohistochemistry using TSA - Part 2 of 4

Simultaneous detection of multiple distinct proteins of interest within a single sample can be achieved with carefully optimized fluorescent multiplex immunohistochemistry (mIHC) using tyramide signal amplification (TSA). This technique utilizes an unconjugated primary antibody specific to the protein of interest, and a primary specific secondary antibody conjugated to horseradish peroxidase (HRP). Detection is achieved with the fluorophore-conjugated HRP substrate, tyramide. When activated, tyramide forms covalent bonds with the tyrosine residues on or near the protein of interest. This permanent deposition allows the primary/secondary antibody pair to be stripped from the sample without disrupting the antigen-associated fluorescence signal. Thus, multiple rounds of staining can be performed in sequence on a given sample, without fear of the crosstalk that would otherwise result from using multiple primary antibodies raised in a single species.

Fluorescent multiplex immunohistochemistry (mIHC) with tyramide signal amplification (TSA) has several advantages over one-color or traditional mIHC.

Benefits of Fluorescent Multiplex Immunohistochemistry using Tyramide Signal Amplification - Part 3 of 4

Both tyramide signal amplification (TSA) and improvements in imaging have enabled the evolution of immunohistochemistry beyond traditional or one-color approaches to fluorescent multiplex immunohistochemistry (mIHC).

Evolution and Future of Multiplex Immunohistochemistry using Tyramide Signal Amplification - Part 4 of 4

Since fluorescent labels were first conjugated to antibodies against targets of interest in the 1940s, a number of landmark developments have enabled the development of immunohistochemistry. These developments include antibody purification and labeling, enzyme digestion and heat-induced approaches to epitope retrieval, signal amplification, and improvements in imaging technologies. 

Bethyl’s Mouse Albumin ELISA kit is a versatile reagent with applications in several molecular biology and biomedical research fields. This is because albumin, which is normally found in the blood, can be used as an indicator of organ function and tissue damage.

Albumin is a Useful Marker of Organ Function and Tissue Damage

Target

Discovered in 2003, B7-H4 is a member of the B7 family of co-stimulatory proteins that includes B7.1 (CD80), B7.2 (CD86), B7-H1 (CD274, PD-L1), B7-H2 (ICOS-L), B7-H3 (CD276) and B7-DC (CD273, PD-L2) that suppresses T cell proliferation and cytokine production. Although treatments targeting CTLA-4 and PD-L1 have had significant success in treating cancer a portion of patients, however, do not respond. This has created a need for alternative immunotherapies targeting different B7 molecules like B7-H4 that may be effective in treating non-responding patients.

Between Yes and No: B7-H4 and T cell Activation

Immunotherapy has become an increasingly important therapeutic option for treating both autoimmune diseases and various cancers. Based on the two-signal hypothesis for T-cell activation, the goal is to either decrease the deleterious effect of an overactive immune system as is the case with autoimmune diseases or the opposite, increase the immune response to enable targeted killing of cancer cells.

T cells with chimeric antigen receptors, more commonly known as CAR-T cells, are used in the immunotherapy of cancer. These cancer-specific T cells are a powerful tool that track down and kill cancer cells.

CAR-T cells for the treatment of blood and solid tumors

T cells with chimeric antigen receptors, more commonly known as CAR-T cells, are used in the immunotherapy of cancer. In CAR-T cell therapy, a patient’s own T cells are taken from their blood and engineered in a laboratory to express an antigen receptor that targets a specific molecule expressed by a patient’s cancer cells. CAR-T cells contain a target-specific extracellular domain fused to the internal domain of CD3-zeta, which may or may not be fused to one or more costimulatory domains. Finally, these cells are transfused back into the patient. These cancer-specific T cells are a powerful tool that track down and kill cancer cells.

Due to the TCR’s extremely sort cytoplasmic tail it is dependent on the CD3 complex for signaling, effectively making the CD3 complex the gatekeeper for T-cell activation. While the CD3 complex consists of multiple components, CD247/CD3Z is arguably the most important due to its role in signal transduction and its potential use as a biomarker for evaluating the status of immune system.

CD247/CD3Z: The Gatekeeper of T-cell Activation

The TCR-CD3 complex plays an essential role in the adaptive immune response. Present on the cell surface, the T-cell receptor (TCR) initiates T-cell activation by recognizing antigens bound to the major histocompatibility complex (MHC) molecule.1 However, due to the TCR’s extremely sort cytoplasmic tail it is dependent on the CD3 complex for signaling, effectively making the CD3 complex the gatekeeper for T-cell activation.2 While the CD3 complex consists of multiple components, CD247/CD3Z is arguably the most important due to its role in signal transduction and its potential use as a biomarker for evaluating the status of immune system.3

Cluster of differentiation 68 (CD68) is a glycoprotein expressed primarily by macrophages that facilitates target identification by binding to tissue- or organ-specific lectins or selectins. CD68 is a macrophage panmarker, enabling identification of the entire macrophage population regardless of phenotype.

CD68 and PD-L1: Better Together?

Cluster of differentiation 68 (CD68) is a glycoprotein expressed primarily by macrophages that facilitates target identification by binding to tissue- or organ-specific lectins or selectins. CD68 is a macrophage panmarker, enabling identification of the entire macrophage population regardless of phenotype. M1 macrophages are proinflammatory and antitumoral, while the M2 phenotype is associated with protumoral and immunosuppressive effects1.

The advent and advancement of immunotherapy is rapidly changing the standard of care of many types of cancer. Since approval of the first modern immunotherapy in 1986, dozens of immunotherapies have been approved for clinical use.

Categories of Cancer Immunotherapy

The advent and advancement of immunotherapy is rapidly changing the standard of care of many types of cancer. Since approval of the first modern immunotherapy in 19861, dozens of immunotherapies have been approved for clinical use2.

Based on the observed association between streptococcal infection of the dermis (erysipelas) and remission of soft tissue sarcoma, the first clinical cancer immunotherapy involved injection of S. pyogenes into patients. Unfortunately, inducing erysipelas proved difficult, as did curing the infection once it was successfully established.

Development of Modern Immuno-oncology

Immune infiltration of human tumors as well as case studies of patients in whom concomitant infection appeared to precipitate remission of otherwise incurable malignancy were reported in the early 1800s.1 Based on the observed association between streptococcal infection of the dermis (erysipelas) and remission of soft tissue sarcoma, the first clinical cancer immunotherapy involved injection of S. pyogenes into patients. Unfortunately, inducing erysipelas proved difficult, as did curing the infection once it was successfully established. As such, the treatment was modified to include both Gram positive and Gram-negative heat-killed, non-infectious bacteria. The resulting immunostimulatory properties of this mixture achieved a remarkable long-term cure rate.2

Body surfaces, cavities, and the lining of hollow organs are comprised of epithelial cells. These cells are characterized by apical-basal polarity and tight cell-cell junctions, and are tightly packed together with little extracellular matrix.

Epithelial–mesenchymal Transition

Body surfaces, cavities, and the lining of hollow organs are comprised of epithelial cells. These cells are characterized by apical-basal polarity and tight cell-cell junctions, and are tightly packed together with little extracellular matrix.1 Due to these cells’ function as coverings and linings, they have one free surface not in contact with other cells, and are attached to underlying connective tissue via noncellular basement membrane.

Regulatory T cells (Treg) are essential for preventing autoimmunity and maintaining peripheral tolerance by downregulating the activation and expansion of effector T cells.

FOXP3: Critical for the Immunosuppressive Function of Regulatory T cells

Regulatory T cells (Treg) are essential for preventing autoimmunity and maintaining peripheral tolerance by downregulating the activation and expansion of effector T cells. Treg are phenotypically diverse and express a number of biomarkers. These biomarkers include cluster of differentiation 4 (CD4) and CD25, while a subset of Treg also express the transcription factor forkhead box P3 (FOXP3). Located on the X chromosome, FOXP3 is crucial for maintaining the functions of Treg.1 FOXP3 plays a critical role in Treg differentiation, and its expression is directly linked to the immunosuppressive activity of these cells.2

Investigation into new potential targets for tumor immunotherapy has been ongoing now for over a decade. One protein that has long been studied in this framework is GITR (TNFRSF18), a member of the TNF superfamily of proteins that is expressed on T cells.

GITR Research in Tumor Immunotherapy

Investigation into new potential targets for tumor immunotherapy has been ongoing now for over a decade. One protein that has long been studied in this framework is GITR (TNFRSF18), a member of the TNF superfamily of proteins that is expressed on T cells. Its ligand, GITRL, is expressed on antigen presenting cells such as dendritic cells. This pathway modulates the T cell immune response through a dual mechanism1 that primarily enhances the proliferation of regulatory T cells (Tregs)2 and also enhances the metabolism of effector T cells3. Because of these dual effects, understanding how best to target GITR in the setting of immunotherapy has remained challenging. Luckily, advances have recently been seen, and the stimulatory effect on regulatory T cells does not appear to be a factor in the cancer setting.

Indoleamine 2,3-dioxigenase 1 (IDO1) is an enzyme that catalyzes the rate limiting step in the conversion of the essential amino acid tryptophan to kynurenine. IDO1 was first identified in the 1950s, but it wasn’t until the 1990s that the immunosuppressive nature of the enzyme was described.

IDO1 - An intracellular target for cancer therapy

Indoleamine 2,3-dioxigenase 1 (IDO1) is an enzyme that catalyzes the rate limiting step in the conversion of the essential amino acid tryptophan to kynurenine. IDO1 was first identified in the 1950s, but it wasn’t until the 1990s that the immunosuppressive nature of the enzyme was described.<sup1,2< sup=""> IDO1 is expressed in both primary and metastatic tumor tissue, and its expression is correlated with advanced disease stage and decreased survival.3,4 Both substrate (tryptophan) and product (kynurenine) of the IDO1-catalyzed reaction can be measured in blood, and an increased ratio of product to substrate has been associated with poor prognosis in breast, cervical, and non-small cell lung cancer, as well as in acute myeloid leukemia and melanoma.4</sup1,2<>

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